Pastuerella haemolytica glycoprotease gene and the purified enzyme

ABSTRACT

A purified DNA molecule encoding a glycoprotease from Pasteurella haemolytica is disclosed. The DNA comprises a sequence of approximately 975 base pairs coding for a glycoprotease having a molecular weight of approximately 35.2 kD. The glycoprotease is specific for cleaving O-glycosylated carbohydrate portions from O-glycoproteins. The glycoprotease has a major cleavage site in glycophorin A between Arg31 and Asp32.

This invention relates to the cloning, sequencing and expression of DNAof a gene for Pasteurella haemolytica glycoprotease. The geneticinformation and expression products may be used in a variety of types ofassays. The purified glycoprotease enzyme may be use in a variety ofchemical and biochemical modifications of glycoproteins.

P. haemolytica is the principal microorganism associated with bovinepneumonic pasteurellosis, a major cause of sickness and death in feedlotcattle in North America. Martin et al. 1980. "Factors associated withmortality in feedlot cattle: The Bruce County beef cattle project."Can.J.Comp.Med.; Yates, W. D. G. 1982. "A review of infectious bovinerhinotricheitis, shipping fever pneumonia and viral-bacterial synergismin respiratory disease of cattle." Can.J.Comp.Med. P. haemolytica hasbeen divided into sixteen serotypes base on soluble or extractablesurface antigens. Biberstein, E. L. 1978. "Biotyping and serotyping ofPasteurella haemolytica." Methods Microbiol. Among the sixteenserotypes, serotype A1 is the predominant microorganism isolated frompneumonic lungs. Smith, P. C. 1983. "Prevalence of Pasteurellahaemolytica in transported calves." Am. J. Vet. Res.; Yates, W. D. G.1982. "A review of infectious bovine rhinotricheitis, shipping feverpneumonia and vital-bacterial synergism in respiratory disease ofcattle." Can. J. Comp. Med. P. haemolytica A1 produces a number ofantigens which are secreted into the culture supernatant during itsgrowth. These antigens include a heat-labile cytotoxin specific forruminant leukocytes, Shewen et al. 1988. "Vaccination of calves withleukotoxic culture supernatant form Pasteurella haemolytica." Can. J.Vet. Med., a serotype-specific outer-membrane protein, Gonzalez et al.1986. "Cloning of Serotype-Specific Antigen form Pasteurella haemolyticaA1." Infect. Immun., a glycoprotease specific for sialoglycoproteins,Otulakowski et al. 1983. "Proteolysis of Sialoglycoprotein byPasteurella haemolytica Cytotoxic Culture Supernatant." Infect. Immun.and neuraminidase Frank, G. H. 198. "Neuraminidase Activity ofPasteurella haemolytica Isolates." Infect. Immun. Vaccination of calveswith bacterial-free culture supernatant form logarithmic phase culturesinduces resistance to experimental challenge. and a vaccine based on theculture supernatant has been developed (Presponse™) Shewen et al. 1988."Efficacy testing a Pasteurella haemolytica extract vaccine." Vet.Med.;Shewen et al. 1988. "Vaccination of calves with leucotoxic culturesupernatant from Pasteurella haemolytica." Can.J.Vet.Med.

The glycoprotease of P. haemolytica A1 is highly specific forO-glycosylated glycoproteins, so that proteins which lack extensiveO-sialoglycopeptides residues are not cleaved by the glycoprotease. Thebest characterized glycoprotein substrate for the glycoprotease isglycophorin A from human erythrocytes. We have found that cleavage bythe enzyme occurs either in situ on the surface of erythrocyte plasmamembrane, or when the substrate glycoprotein is in solution. This enzymeis a neutral metallo-protease and is non-toxic to cultured mammaliancells including bovine pulmonary macrophages, bovine endothelial cellsand erythrocytes Otulakowski et al. 1983. "Proteolysis ofSialoglycoprotein by Pasteurella haemolytica Cytotoxic CultureSupernatant." Infect. Immun. The role of the glycoprotease inpathogenesis and in the induction of an immune response is unknown. Ahomogeneous enzyme preparation for the glycoprotease is difficult toisolate by conventional biochemical techniques.

We have discovered and cloned a gene for the P. haemolyticaglycoprotease. We have cloned, sequenced and expressed the gene. Theexpression product of the gene can be isolated to provide theglycoprotease in homogeneous form. The enzyme has restricted substratespecificity, which has a variety of chemical and biochemical uses inmodifying glycoproteins. Such uses include, characterization ofglycoproteins through specific sites of cleavage by the glycoprotease,the glycoprotease may be used to dissect components of the immuneresponse by the select cleavage of surface molecules on immune cells.The glycoprotease may also be used to facilitate immunopurification ofvarious types of cells by cleaving surface glycoproteins.

Accordingly, aspects of the invention are as follows:

1) The gene codes for the P. haemolytica enzyme, which has uniquesubstrate specificity. As recorded previously, it cleaves the cellsurface O-sialoglycoprotein glycophorin A, either in situ on the surfaceof human erythrocytes, or when the protein is free in solution. Themajor site of cleavage is the amide bond between the Arginine 31 and theAspartate 32 residues. Minor site cleavage appear to be at theAla7-Met8, Try34-Ala35, Glu60-Arg61, Ala65-His66 and His67-Phe68 amidebonds.

2) The enzyme cleaves other glycoproteins. The enzyme cleaves the humancell surface glycoproteins CD34 (primitive bone marrow stem cellantigen), CD43 (leukosialin/sialophorin), CD44 (hyaluronate receptor).and CD45 (leukocyte common antigen). Other cell surface glycoproteins ofhumans and other organisms are good substrates for the enzyme action.

3) The gene can be used to make recombinant enzyme free from otherproteins of P. haemolytica, and this enzyme can be used to generatespecific polyclonal or monoclonal antibodies. The antibodies are usefulin the screening of other organisms which may make similar enzyme. Theantibodies can be used to neutralize the enzyme action, to determine theroles of the enzyme in disease processes where the bacterium P.haemolytica is known to be involved, and to screen for involvement ofthis bacterium in diagnosis of infection.

4) The gene sequence may be used to make probes, DNA polynucleotides,which will hybridize with native DNA in other bacteria and will detectthe occurrence of this or similar (homologous) genes in other bacteria.

5) The gene sequence has revealed extensive homology with an unknowngene of Escherichia coli, a gene called Orfx. The E. coli gene productmay be a proteolytic enzyme with some resemblances to the P. haemolyticaglycoprotease.

6) The enzyme can be used to generate novel biological products by thecleavage of O-sialoglycoproteins of natural origin. The cleavage of thehuman platelet and endothelial cell surface glycoprotein thrombomodulin,by the P. haemolytica glycoprotease, may be used to generate a solubleanticoagulant with potential uses in the treatment of blood clottingdisorders, including DIC (disseminated intravascular coagulopathy).

Various aspects of the invention are as follows:

1) A purified DNA molecule comprises a DNA sequence of approximately 975bp coding for a glycoprotease having a molecular weight of approximately35.2 kD, the DNA molecule having a restriction map of FIG. 2B.

2) A purified DNA molecule comprises the DNA sequence of FIG. 4 (SEQ IDNO:1) for bp positions 1 through 975.

3) A purified DNA molecule comprises at least 12 nucleotides selectedfrom the group of nucleotides represented by bp positions 1 through 975of FIG. 4 (SEQ ID NO:1).

4) A purified DNA molecule comprises a DNA sequence encoding aglycoprotease having an amino acid sequence of FIG. 4 (SEQ ID NO:1or 2).

5) A purified DNA molecule comprises at least 18 nucleotides encoding afragment of a glycoprotease amino acid sequence, the fragment beingselected from at least 6 corresponding sequential amino acid residues ofthe amino acid sequence of FIG. 4 (SEQ ID NO:1or 2).

6) A purified DNA probe comprises a DNA sequence of item 3.

7) A recombinant cloning vector comprises a DNA molecule of items 1, 2,3, or 4.

8) A host is transformed with a recombinant cloning vector comprises aDNA molecule of items 1, 2, 3 or 4.

9) A method for screening a biological sample to determine presence of agene encoding a glycoprotease of FIG. 4 (SEQ ID NO:1), comprises:

i) providing a biological sample derived from a source suspected of aserotype of P. haemolytica which produces the glycoprotease,

ii) conducting a biological assay to determine presence in thebiological sample of at least a member selected from the groupconsisting of:

a) glycoprotease gene sequence of FIG. 4 (SEQ ID NO:1), and

b) glycoprotease amino acid sequence of FIG. 4 (SEQ ID NO:1or 2).

10) A purified glycoprotease protein produced by a host and isolatedfrom culture of the selected cells, the protein having the amino acidsequence of FIG. 4 (SEQ ID NO:1or 2).

11) A purified glycoprotease protein fragment comprises at least sixamino acid residues of the sequence of FIG. 4 (SEQ ID NO:1or 2).

12) An antibody specific for the glycoprotease.

13) A glycoprotease for cleaving glycoproteins which have portions richin O-glycosylated carbohydrates, said glycoprotease having highlyrestricted specificity for cleaving solely said O-glycosylatedcarbohydrates portions from said glycoproteins, said glycoprotease beingcharacterized by:

i) a molecular weight of 35.2 kD,

ii) cleaves glycophorin A at a major site between Arg 31-Asp 32 and atminor sites at least between Try 34-Ala 35, Glu 60 -Arg 61 and Ala65-His 66,

iii) a protein secreted from cultured P. haemolytica cells of selectedserotypes 1, 2, 5, 6, 7, 8, 9 and 12, and

iv) stable protease activity at pH 7.0-7.5.

14) A method for enzymatically cleaving only O-glycosylated carbohydrateportions from glycoproteins on cells surfaces, the method comprisestreating cells having the glycoproteins rich in the O-glycosylatedportions with the glycoprotease.

15) A cell having solely O-sialoglycosylated carbohydrate proteinportions of glycoproteins in its cell wall enzymatically cleavedtherefrom by treatment with the glycoproteins.

16) A vaccine comprises glycoprotease of item 14 and a pharmaceuticallyacceptable carrier for vaccine administration.

Aspects of the invention are demonstrated with respect to the drawingswherein:

FIG. 1. Glycoprotease activity in E. coli recombinant clones. Lanes: a,¹²⁵ I-glycophorin A only; b, E. coli carrying pBR322; c, E. colicarrying pPH1; d, E. coli carrying pPHS; e through k are the other E.coli clones of the genomic library. (GPA)₂, glycophorin A dimer; GPA,glycophorin A monomer.

FIG. 2. Restrictions maps of plasmids used in this study. (A)Restriction map of insert DNA in pPH1 and pPH8. The arrows indicate thedirection and extent of DNA sequenced. (B) Subcloning of 3.3-kpbBamHi-BgIII fragment from pPH1 into pTTQ19 in either orientation. p,direction of expression from the tac promoter in pPH1.1 and pPH1.2; darkbars with ΔA and ΔE, internal fragments deleted in the plasmids pPH1.2Aand pPH1.1E, respectively; open bar, open reading frame coding for gcpas deduced from the DNA sequence. Abbreviations: A, AvaI; B, BamHI; Bg,BgIII; B/S, BamHI-Sau3A junction; E, EcoRI; H, HindIII; X, Xbal.

FIG. 3. Maxicell labelling of the plasmid-encoded proteins.Autoradiogram of 15% SDS-PAGE containing [³ S]methionine-labelledproteins expressed in E. coli carrying pBR322 (lane a), pPH1 (lane b),pPH1.1 (lane c), pPH1.2 (lane d), pPH1.1A (lane e), pPH1.1E (lane f),and pTTQ19 (lane g). The β-lactamase of pBR322 and the Lac repressorprotein of pTTQ19 are indicated by Bla and I, respectively. Molecularsize standards, starting from the top, represent 92.5, 66, 43, 31 and 21kDa. The solid circles in the gel show the position of the 53-kDaprotein corresponding to the glycoprotease.

FIG. 4. Nucleotide sequence (SEQ ID NO:1) of insert DNA between theBamHI and EcoRI sites in pPH1.1. The numbers above each line refer tonucleotide positions, which are arbitrarily numbered from -140 at theBamHI site to 1170 at the EcoRI site. The predicted amino acid sequencefor the glycoprotease is shown beneath the DNA sequence (SEQ ID NO:2).The inverted repeats downstream from the gcp translational stop codonare underlined with arrows. Abbreviations: B, BamHI; E, EcoRI.

FIG. 5. Hydropathy profile of the glycoprotease protein. The method usedwas that of Klein, P., M. Kanehisa and C. DeLisi. 1985. The Detectionand Classification of Membrane-Spanning Proteins. Biochem. Biophys.Acta. 815:468-476. The vertical axis gives the scale of the hydrophobic(positive) and hydrophilic (negative) values established for each windowof nine residues. The horizontal axis gives the scale for the aminoacids in protein.

FIG. 6 Homology between the glycoprotease (SEQ ID NO:2) and OrfX (SEQ IDNO:3). The segmented vertical lines show the location of identicalresidues. The segmented horizontal lines represent repressor breaksintroduced to maximize homology. Potential zinc ligand of theglycoprotease and OrfX at His-111 and His-115 is underlined.

FIG. 7. High-level expression and secretion of the glycoprotease. (A)Total cellular proteins expressed from cultures of E. coli HB101carrying pTTQ18 (lane 1), uninduced pGP1 (lane 2), pTTQ18 (lane 3), andpGP1 induced with IPTG (lane 4) separated by 15% SDS-PAGE and stainedwith Coomassie blue. The glycoprotease protein is indicated by anarrowhead. (B) Periplasmic fractions after osmotic shock treatments toE. coli HB101 carrying induced pGP1. Lane 1, Cytoplasmic fraction; lane2, periplasmic fraction. Molecular size standards are in kilodaltons.

FIG. 8. Enzyme activity of the highly expressed glycoprotease in E. coliHB101. Lane a, ¹²⁵ I-glycophorin A only; lane b, negative control,protein extract from E. coli carrying pTTQ18; lane c, positive control,concentrated culture supernatant of P. haemolytica A1; lane d, proteinextract from E. coli carrying. pGP1 after IPTG inductions. (GPA)₂,glycophorin A dimer; GPA, glycophorin A monomer.

FIG. 9. Autoradiographs of SDS-PAGE gels showing hydrolysis of [¹²⁵I]-glycophorin A. A: Lane (a) [125I]-glycophorin A (20 μCi per mg), 1.7per lane; lane (b) glycophorin A incubated with P. haemolytica A1 pH 4.5fraction, 0.3 mg protein in 67 μl 0.1M HEPES buffer, pH 7.4, for 30 min;lane (c) 1.7 μg [¹²⁵ I]-glycophorin A after desialylation withneuraminidase; lane (d) desialated glycophorin A incubated with P.haemolytica A1 fraction for 30 min; lane (e) glycophorin A incubatedwith P. haemolytica A1 fraction in the presence of 100 mM EDTA; lane (f)glycophorin A incubated with P. haemolytica A1 fraction, treated with100 mM EDTA and then dialyzed overnight prior to incubation withsubstrate. B: Hydrolysis of 1.7 μg [¹²⁵ I]-glycophorin A by P.haemolytica A1 culture supernatants (0.3 mg protein, pH 4.5 fraction) inHEPES buffer as above, over 80 min. The incubation are: lane (a) noincubation; lane (b) 5 min; lane (c) 10 min; lane (d) 20 min; lane (e)40 min; lane (f) 80 min. A₂ is glycophorin A dimer, A is glycophorin Amonomer.

FIG. 10. Gel filtration HPLC separation of P. haemolytica A1 culturesupernatant proteins. Serum-free culture supernatant was concentrated byultrafiltration, with a 10 kD membrane pore size, to yield 100 μg ofprotein, which was chromatographed in Tris-HCl buffer, 0.1M pH 7.4,through two TSK G4000-SW molecular sieving columns 60 cm×0.75 cm,coupled in series, separation range 5-1000 kD. Elution was at 1 ml permin and six ml fractions were collected for glycoproteinase activitydetermination by the cleavage of [¹²⁵ I]-glycophorin A (shaded bars) andneuraminidase activity determination by the cleavage of the fluorogenicsubstrate (open bars), with monitoring of protein absorbance at 214 nm(solid line).

FIG. 11. (A) Anion exchange HPLC of fraction 6, from TSK columns.Fraction 6 (six mil) from the gel exclusion HPLC shown in FIG. 10, wasconcentrated by ultrafiltration, 10 kD membrane pore size, to yield 28.8μg of protein. Twenty μg of this protein was chromatographed on a Mono QHR 5/5 column and 1.0 ml fractions were eluted at 0.5 ml/min in Tris-HCLbuffer, 0.1M pH 7.4, by a gradient of 0.0 to 0.5M NaCl. (B) SDS-PAGEanalysis of fractions eluted from Mono Q column. (Lane 1) Molecularweight markers; Lane (2) Total proteins in the starting material,serum-free culture supernatant; (Lane 3) Fraction 24; (Lane 4) 1.7 μg[¹²⁵ I]-glycophorin A substrate in 67 μl HEPES buffer pH 7.4; (Lane 5)glycophorin A as in lane 4, but incubated with 133 μl fraction 24 forone hour. Lanes 1-3 were visualized by silver-staining, and lanes 4 and5 by autoradiography.

The discovery of the gene sequence (SEQ ID NO:1) which encodes for theP. haemolytica glycoprotease provides a number of significant advantagesin the detection, molecular assays, protein mechanisms and medical carerelated to diseases caused by P. haemolytica infection and the uses ofenzyme in cleaving glycoproteins and in particular O-sialoglycoproteins.Various DNA probes can be developed from the sequence (SEQ ID NO:1)described for purposes of detecting complementary DNA sequences invarious types of biological materials including living cells. Portionsof or the entire sequence (SEQ ID NO:1) may be expressed in anappropriate expression vehicle, which may be bacterial cells, viruses,plant, animal and in particular mammalian cells. The glycoprotease asproduced by the expression of the DNA, or fragments of the enzyme asproduced by corresponding fragments of the DNA may be used to performvarious cleaving operations on molecules or other biological matterincluding living cells which have surface proteins includingO-sialoglycoproteins. Expression of the selected DNA sequence in atransformed expression system provides purified enzyme, although it isappreciated the enzyme may be purified from culture of the selected P.haemolytica serotype. The produced protein may be used to developvarious poly and monoclonal antibodies which are effective in diagnosisand various types of assays as well as potential uses in passivevaccines. The protein may also be used directly in various forms ofanimal vaccines in the treatment of P. haemolytica infections and inparticular in combination with the vaccines disclosed in applicant'sco-pending applications Ser. No. 786,662,720,332 and 462,929. There areof course other uses of the enzyme which have already been described orshall be described in the following description of this invention.

The cloning of the glycoprotease gene may be achieved in accordance withthe following manner, employed by the inventors in discovering the gene.

The lysates from twenty-seven recombinant clones in E. coli were assayedfor glycoprotease activity. The results in FIG. 1 shows anautoradiograph of the hydrolysis of radio-iodinated glycophorin Aincubated with lysate from clone pPH1. A negative control of the pBR322transformed E. coli shows no hydrolysis, and a positive control of P.haemolytica A1 culture supernatant shows complete hydrolysis. Prolongedautoradiography of the gel also showed weak hydrolysis of ¹²⁵S-glycophorin A by the lysate from another clone pPHS. The activity inpPH1 and pPH8 was 50% and 10% respectively of the glycoprotease activityof the culture supernatant sample from P. haemolytica A1. Restrictionendonuclease analysis of the recombinant plasmids pPH1 and pPH8 showedthat both plasmids contain an identical insert (FIG. 2), and pPH1 waschosen for further studies.

The plasmid pPH1 was transformed in E. coli CSR603 and theplasmid-encoded proteins examined by in vivo ³⁵ S-labelling. The resultsshow that an additional protein of about 35 KD was expressed from pPH1(FIG. 3). This gives an estimate of the size of the glycoprotease and isconsistent with the estimated size of the enzyme, in SDS-PAGE analysisof extracts of P. haemolytica A1 culture supernatant. To locate thecoding region of the glycoprotease gene on pPH1, the 3.3 kbp BamHI-BglIIfragment was subcloned into the expression vector pTTQ19 to produce thesubclones pPH1.1 and pPH1.2 which carry the insert DNA in oppositeorientations (FIG. 2B). The plasmid-encoded proteins from pPH1.1 andpPH1.2 were examined in the E. coli maxi-cell system and the results areshown in FIG. 3. Only plasmid pPH1.1 expressed a plasmid-encoded proteinidentical in size to the cloned protein expressed from pPH1, suggestingthat this plasmid encodes the correct orientation for expression. Twosubclones were constructed from pPH1.1 by deleting selective internalfragments to yield the constructs pPH1.1A and pPH1.1E (FIG. 2C).Subclone pPH1.1A (constructed by partial digestion of pPH1.1 with AvaIand relegation) did not express the 35 kD protein whereas pPH1.1E stillexpresses the protein similar to that observed in pPH1 and pPH1.1 (FIG.3). This showed that AvaI site is located within the glycoprotease geneand the EcoRI site may be close to the end of the gene. Based on thesize of the glycoprotease expressed from the maxi-cell analysis, a DNAfragment of about 1 kbp is required to encode for this 35 kD protein andthis gene must be located within the 2.3 kbp BamHI-HindIII fragment inthe orientation depicted in pPH1.1.

The complete nucleotide sequence of the 2.3 kbp BamHI-HindIII fragmentwas determined and the 1.3 kbp sequence between the BamHI and EcoRI siteis presented in FIG. 4 (SEQ ID NO:1). More than 80% of both strands ofthe DNA were sequenced either directly, or by the use of overlappingdeletions of the cloned DNA, in the phage vectors M13 mp18 or M13 mp19.Most regions were sequenced at least three times independently inaccordance with the procedure. Analysis of the DNA sequence revealed onelarge open reading frame expressing in the direction anticipated fromthe maxi-cell labelling experiments (FIG. 2). The open reading framecovers a length of 975 nucleotides and encodes for 325 amino acids withpredicted molecular weight of 35.2 kD. These estimates are in agreementwith size of the protein expressed in the maxi-cell labellingexperiments. For purposes of further discussions, the gene encoding theglycoprotease is designated gcp.

The cloned glycoprotease was prepared from plasmid pGP1 (see below) ashereinafter described by SDS-PAGE and electroblotted onto PVDF membrane.The membrane region containing the glycoprotease was excised and theN-terminal amino acid sequence (SEQ ID NO:2) was determined. The resultsfrom the first eight cycles are identical to the first eight amino acidspredicted from the nucleotide sequence of gcp and confirms theassignment of the reading frame for the glycoprotease.

An examination of the nucleotide sequence (SEQ ID NO:1) upstream fromgcp did not reveal features similar to the promoter commonly found in E.coli (FIG. 4). Neither the consensus promoter sequences TATAAT nor theconsensus RNA polymerase binding site TTGACA have been identified.Further, a putative ribosome-binding site immediately preceding the ATGinitiation codon of gpc is also absent (FIG. 4). It is possible that thegcp promoter is not readily recognized in E. coli and this may explainthe poor expression of glycoprotease activity in the initial clones pPH1and pPH8. On the other hand, downstream from the termination codon ofgcp, a mRNA structure consisting of a 14-bp stem and loop structuresimilar to the rho independent transcriptional signal of E. coli couldbe identified (FIG. 4). In two other loci sequences from P. haemolytica,sequences similar to the E. coli promoters can be identified, Lo et al.1987. "Nucleotide Sequence of the Leucotoxin Genes of Pasteurellahaemolytica A1." Infect. Immun.; Strathdee et al. 1989. "Cloning,nucleotide sequence, and characterization of genes encoding thesecretion function of the Pasteurella haemolytica leukotoxindeterminant." J. Bacteriol. As is understood, it is possible thatdifferent types of promoters under different regulatory systems areutilized in P. haemolytica.

The predicted amino acid sequence (SEQ ID NO:2) of the glycoprotease wasanalyzed for its hydrophobicity and potential membrane spanning regions.FIG. 5 shows a hydropathy plot of the glycoprotease analyzed by the SOAPprogram (Klein et al supra). The analysis classified the glycoproteaseas a potential peripheral membrane protein but not as an integralmembrane protein with transmembrane domains. This is consistent with theglycoprotease found among the secreted products of P. haemolytica A1.

A search of the nucleotide sequence of gcp with databanks such asGenBank showed extensive homology with the DNA region upstream from theE. coli rpsU-dnaG-rpoD macromolecular-synthesis operon. In particular,gcp (SEQ ID NO1) is almost identical to an identified gene designatedOrfX (SEQ ID NO:3) in that region Nesin et al. 1987. "Possible new genesrevealed by molecular analysis of 5-kb Escherichia coli chromosomalregion 5 to the rpsU-dnaG-rpoD macromolecular-synthesis operon." Gene. Acomparison of the predicted amino acids of glycoprotease (SEQ ID NO:1)and OrfX (SEQ ID NO:3) is shown in FIG. 6. Almost 76% of the amino acidsof glycoprotease are identical to those of OrfX, suggesting that the twoproteins have similar functions. On the other hand, the codon usage inthe two genes and the flanking nucleotide sequences are very dissimilar.Little is known about the function of the protein encoded by orfX exceptthat it may be involved in regulation of expression of therpsU-dnaG-rpoD operon, Nesin et al. 1987 (supra). However, based on thisinvention it is most likely that a proteolytic activity is associatedwith the OrfX protein, which may be part of the regulation ofmacromolecule biosynthesis.

The 2.3 kbp BamHI-HindIII fragment was then subcloned into the BamHI andHindIII site of a high expression vector pTTQ18 to produce plasmid pGP1.This placed the initiation codon of gcp at an appropriate distance fromthe tac promoter to allow high expression of the glycoprotease. FIG. 7shows expression of the P. haemolytica A1 glycoprotease in E. colicarrying pGP1, upon induction with IPTG (isopropyl-β-D-thiogalactoside).No glycoprotease could be detected in E. coli carrying only PTTQ18, norin uninduced cultures (FIG. 7). The same protein preparations were alsoassayed for glycoprotease activity and the results in FIG. 8 showed thatE. coli carrying pGP1 expressed enzyme activity identical to that foundin the P. haemolytica A1 culture supernatant.

The overexpressed 35-kDa protein isolated by SDS-PAGE from E. colilysates containing the glycoprotease activity was used to raisepolyclonal antibody in rabbits. This antiserum was used to neutralizethe glycoprotease activity in culture supernatants of P. haemolytica A1.Under conditions in which the P. haemolytica glycoprotease degraded 16.8μg of glycophorin A in 30 min, when rabbit anti-glycoprotease antiserumwas added at titers of 1/4, 1/8, 1/16 and 1/32, degradation ofglycophorin A dropped to 4.0, 8.9, 8.4, and 11.5 μg of glycophorin A per30 min, respectively. Control antisera against 35-kDa bands from lysatesof plasmid-transformed E. coli which lacked the pGP1 gene showed noinhibition of the P. haemolytica glycoprotease.

The glycoprotease of P. haemolytica A1 is normally secreted in theculture medium, but its mechanism of secretion is not known. After IPTGinduction of E. coli carrying pGP1, the cells were subjected to osmoticshock treatment to determine the location of the glycoprotease. Theglycoprotease is located in the periplasmic fraction of the E. coliclone. Only traces of the glycoprotease protein were detected in thecellular fraction. An examination of the DNA-derived N-terminal aminoacid sequence of the glycoprotease (SEQ ID NO:2) showed the absence of aconventional signal peptide sequence. Furthermore, N-terminal amino acidsequence (SEQ ID NO:2) analysis of the glycoprotease expressed in E.coli showed that there is no cleavage of amino acids from the N-terminalregion during export of the glycoprotease to the periplasm in E. coli.This suggests that there may be a different mechanism involved in thesecretion of the glycoprotease.

The cloning and sequencing data show that the P. haemolytica A1glycoprotease is a protein of 35.2 kD. The enzyme has a predicted pI of5.2 which is consistent with our finding that the enzyme activity isprecipitated from culture supernatants by lowering the pH to 4.5.

In the initial stages of developing expression of the gene, only lowlevel of glycoprotease activity was expressed in E. coli transformed bypPH1 (or pPHS). This was most likely due to the inefficient activity ofthe P. haemolytica A1 gcp promoter in E. coli or to incompleteprocessing of the enzyme, or to instability of the enzyme in E. coli.Upon subcloning of the appropriate DNA fragment into pTTQ18, the levelof glycoprotease activity in E. coli was increased, however, thespecific activity of the enzyme preparation was less than that observedin serum-free culture supernatants of P. haemolytica A1. The lowerspecific activity of the recombinant product was probably due to a lackof post-translational processing of the cloned glycoprotease gene in E.coli.

The leukotoxin is another secreted protein of P. haemolytica A1, forwhich the nucleotide sequence and the regulation of leukotoxinexpression has been described, Lo et al. 1987. "Nucleotide Sequence ofthe Leucotoxin Genes of Pasteurella haemolytica A1. "Infect. Immun.;Strathdee et al. 1989a, "Cloning, nucleotide sequence, andcharacterization of genes encoding the secretion function of thePasteurella haemolytica leucotoxin determinant." J. Bacteriol; Strathdeeet al 1989b. The leukotoxin determinant is composed of four contiguousgenes lktCABD encoded on the same DNA strand where LktA is thestructural gene for the leukotoxin (LktA), while proteins encoded bylktC functions in the activation of leukotoxin (LktA), while proteinsencoded by lktB and lktD are involved in the secretion of leucotoxin. Itpossible that glycoprotease has a similar mode of activation as theleukotoxin, which could explain the lower specific activity of theenzyme expressed in E. coli. An examination of the amino terminus of theglycoprotease from pGP1 shows no pattern similar to the conventionalsignal sequences predicted for a number of secreted proteinscharacterized Michaelis et al. 1982. "Mechanism of incorporation of cellenvelope proteins in Escherichia coli." Ann. Rev.; Silhavy et al. 1988."Mechanism of protein localization." Microbiol. Rev.; Von Heijne, G.1983. "Patterns of amino acids near signal sequence cleavage sites."Eur. J. Biochem. Since the glycoprotease is normally secreted from P.haemolytica A1, an alternative secretory mechanism not involving anamino-terminus signal may be utilized, as reported for the leukotoxin,Strathdee et al. 1989. "Cloning, nucleotide sequence, andcharacterization of genes encoding the secretion function of thePasteurella haemolytica leukotoxin determinant". J. Bacteriol.

It is therefore appreciated that with the variety of expression vehiclesand expression systems and with the information provided by the genesequence and the analysis of the corresponding protein sequence for theglycoprotease, manufacture of purified glycoprotease can be realized bythose skilled in the art.

The glycoprotease of P. haemolytica A1 appears to have some similarityof action with other neutral metallo-protease of bacteria, such asthermolysin Nakahama et al. 1986. "Cloning and sequencing of Serratiaprotease gene." Nucleic Acids Res, but there is no major sequencehomology with these enzymes, except for the presence of a potential zincbinding site (FIG. 6). The unusual substrate specificity for theglycoprotease, namely its specificity for O-sialoglycosylated proteinsand the lack of homology with other known protease suggest that it is amember of a new enzyme class.

Prior to our discovery, little was known about the relationship betweenthe glycoprotease of P. haemolytica A1 and other bacterial proteases.High level expression of pGP1 in E. coli upon induction with IPTG allowslarge scale preparation of the glycoprotease free from P. haemolytica A1proteins. Such preparation can be used for detailed studies on theactivities an immunological properties of the enzyme. Polyclonalantibodies are readily prepared in rabbits using the cloned enzyme. Thisis an important reagent for further characterization of biochemical andbiological properties of the glycoprotease.

It is appreciated that with the protein sequence information for theglycoprotease, selected functional regions of the protein may be used inraising antibodies or the entire protein may be used in raisingantibodies. In accordance with standard techniques the above-mentionedpolyclonal antibodies may be developed, although it is understood thatfor particular applications monoclonal antibodies may be developed fordiagnostic uses and the like. It is also appreciated that in the processof developing various forms of antibodies protein sequences normally of6 residues or more would be selected.

It is also appreciated that the DNA sequence (SEQ ID NO:1) provides thenecessary information to develop a variety of DNA probes which can beused in corresponding diagnosis. The probes can be developed fromselected portions of the DNA sequence (SEQ ID NO:1) for purposes ofdetermining the presence of certain functional portions of the sequence.It is generally understood that for purposes of DNA hybridizationpreferably 14 to 18 base pairs are selected. Such probes may be used toscreen serotypes of P. haemolytica for the occurrence of theglycoprotease gene such as discussed in Abdullah et al. December 1990."Distribution of glycoprotease activity and the glycoprotease gene amongserotypes of Pasteurella haemolytica." Biochemical Society Transactions.

The glycoprotease was found to be unstable when isolated by HPLC fromserum-free culture supernatants. However, alternative procedures forisolation and purification from culture are provided hereinafter. Thisis in marked contrast to the remarkable stability of the enzyme activityin freeze-dried pH 4.5 precipitates of culture supernatant, in whichactivity is maintained for many months at room temperature. Theincreased protein concentrations of the recombinant gene productexpressed in highly active expression vectors should overcome theliability of the enzyme at low protein concentrations. The glycoproteaseis only a minor protein component of the culture supernatant of P.haemolytica even in bacteria grown in serum-free media. Consequently ithas been difficult to isolate a homogeneous preparation of theglycoprotease, except by laborious chromatographic methods. The recentidentification of the recombinant glycoprotease as a 35 kD³⁵ S-labelledband on SDS-PAGE, the high level of expression of this product, and therecognition that the recombinant product is located within theperiplasmic space of E. coli HB101 enables one to obtain large amountsof highly purified product.

The best understood substrate for the glycoprotease other than theerythrocyte sialoglycoprotein is glycophorin, A Udoh et al. 1986. Univ.of Guelph. None of thirty proteins and glycoproteins tested previouslywas cleaved by the enzyme. When glycoproteins from various sources wereradiolabelled with ¹²⁵ I-iodine and incubated with partially-purifiedenzyme, no hydrolysis of these substrates could be detected, by SDS-PAGEand autoradiography. No hydrolysis was seen for human immunoglobulin A1(IgA1) or human immunoglobulin A2 (IgA2), so that theglycophorin-degrading enzyme is not identical to IgA protease, amicrobial neutral metalloprotease Plaut et al. 1983. Annu. Rev.Microbiol. Similar procedures showed that the P. haemolytica proteasedoes not degrade [¹²⁵ I]-labelled bovine α-1 acid glycoprotein, bovineβ-lactalbumin, or hen ovalbumin. The [¹²⁵ I]-labelled proteins, bovineserum albumin, glyceraldehyde-3-phosphate dehydrogenase, soybean trypsininhibitor, bovine carbonic anhydrase, trypsinogen and chymotrypsinogenwere not cleaved by the P. haemolytica protease. Other proteins weretested as substrates by incubation with active enzyme fractions, andenzyme action was monitored by SDS-PAGE and Coomassie blue staining ofthe substrate products. The enzyme did not hydrolyze insulin chain A,insulin chain B, or cytochrome c. Partially purified enzyme preparationswith high activity against glycophorin were inactive in cleavage ofdye-casein conjugates (Azocasein) or dye-collagen conjugates (Azocoll).Thus the weak casein-degrading activity reported in culture supernatantsof P. haemolytica Otulakowski et al. 1983. Infect. Immun. was not foundin the glycoproteinase-enriched extracts used here.

The removal of sialyl residues from glycophorin, by treatment withneuraminidase destroys the susceptibility of the glycoprotein tohydrolysis by the enzyme. The high degree of specificity for an O-linkedsialoglycoprotein, of a type commonly found on mammalian cell surfaces,is a unique property of this enzyme. Our results suggest that a majorcleavage of glycophorin A occurs at the Arg31-Asp32 and this has beenconfirmed by N-terminal analysis of the one product, but there are othersites of cleavage. These other sites are in the glycosylated N-terminalregion close to the O-linked sialoglycosylated residues which were notedabove.

The glycoprotease activity is stable at pH 7.0-7.5, but is destroyedrapidly above pH 8.0. The enzyme can be inhibited by prolonged treatment(24h) with phenanthroline, so that it was tentatively classified as aneutral metallo-protease Otulakowski et al. 1983. Infect. Immun. EDTA isa poor inhibitor, unless the enzyme is prepared and assayed inserum-free media. Some inhibitions are given by millimolarconcentrations of the acidic amino acids aspartate and glutamate but notby their amide analogues. The anions citrate, ascorbate and phosphategive similar inhibition, but sialate does not. The inhibition probablyarises from competition between anions and the sialoglycoproteinsubstrates, in binding to the enzyme. It is unlikely that the inhibitionby these anions is due to chelation of essential divalent metal cationsby the inhibitor, since EDTA is a relatively weak inhibitor compared tocitrate, ascorbate, aspartate, glutamate, and phosphate. TheEDTA-inhibited P. haemolytica glycoprotease activity could not bereactivated by the addition of metal ions. Phosphoramidon[N-(β-L-rhamnopyranosyl-oxyhydroxy-phosphinyl-L-leucyl-L-tryptophan] isa potent inhibitor of neutral metallo-proteases such as thermolysinMalfroy et al. 1985. Biophys. Res. Commun. but without significanteffect on the P. haemolytica protease. Lack of inhibition byphosphoramidon is consistent with the inability of the enzyme tohydrolyze the thermolysin substrate, furoylacryloylglycylleucinamideOtulakowski et al. 1983. Infect. Immun. The protease is not asheatstable as is thermolysin. There are several O-sialoglycoproteins,found on human cell surfaces, which are substrates for the P.haemolytica glycoprotease. These new substrates are well characterizedleukocyte antigens with diverse functions. The enzyme thus provides anew tool with which to study the structure-function relationships forsome cell surface antigens.

It is appreciated that the glycoprotease may also be produced byisolating and purifying the enzyme from a culture of a suitable strainof P. haemolytica. There are several serotypes which produce theglycoprotease. As we reported in (Abdullah et al. supra), P. haemolyticaserotypes 1, 2, 5, 6, 7, 8, 9 and 12 secrete the glycoprotease. Forpurposes of understanding such isolation and purification steps andfurther understanding the purified glycoprotease, the followingdiscussion is provided.

Protease purification:

Culture supernatants from a majority of the serotypes of P. haemolyticaA1 contain a neutral protease (Otulaklowski, G. L. P. E. Shewen, A. E.Udoh, A. Mellors, and B. N. Wilkie. 1983. Proteolysis ofSialoglycoprotein by Pasteurella Haemolytica Cytotoxin CultureSupernatant. Infect. Immun. 42:64-70. FIG. 9 (A) shows that the culturesupernatant from P. haemolytica A1 biotype A, serotype 1, contains anenzyme activity that cleaves human [¹²⁵ I]-labelled glycophorin A, asrevealed by the disappearance of dimeric glycophorin A and monomericglycophorin A (lane a), to yield corresponding dimeric and monomericproducts (lane b). Glycophorin A is the major sialoglycopeptide of thehuman erythrocyte membrane (Marchesi, V. T., H. Furthmayr, and M.Tomita. 1976. The Red Cell Membrane. Ann. Rev. Biochem. 45:667-698 andsimilar O-linked sialoglycopeptides are found on the cell surface oflymphoid cells (Fukuda, M., and S. R. Carlsson. 1986. Leukosialin, AMajor Sialoglycoprotein on Human Leukocytes as Differentiation Antigens,Med. Biol. 64:335-343, Remold-O-Donnell, E., A. E. Davis III, D. Kenney,K. R. Bhaskar, and F. S. Rosen. 1986. Purification and ChemicalComposition of gpL115, The Human Lymphocyte Surface Sialoglycophorinthat is Defective in WiskottAldrich Syndrome. J. Biol. Chem.261:7526-7530. When glycophorin A was extensively desialated bytreatment with Clostridium perfringens neuraminidase, as monitored byWarren's assay (Warren, L. 1959. The Thiobarbituric acid assay of sialicacids. J. Biol. Chem. 234:1971-1975), little or no hydrolysis of thedesialated dimeric or monomeric glycophorin, by the P. haemolyticaprotease, could be seen (lane d of FIG. 9(A)). The hydrolysis ofglycophorin A can be inhibition by the presence of 100 mM EDTA, but thisinhibition by high concentration of EDTA was removed by dialysis of theEDTA-inhibited enzyme (lane f FIG. 9(A)). FIG. 9(B) shows that if theincubation of the glycophorin A with P. haemolytica pH 4.5 fractions wasprolonged, then total hydrolysis of the two forms of glycophorin A wasobserved.

To facilitate the purification of the enzyme, glycophorin was tritiatedin situ in human erythrocyte ghost membranes by periodate oxidation andsodium-[³ H]-borohydride reduction. The [3H]-labelled erythrocyte ghostswere used to assay the enzyme activity during the purification of theproteolytic activity. Soluble radiolabelled glycophorin cannot bereadily used in a simple enzyme assay, because neither the substrate northe products can be precipitated. However, radiolabelled glycophorin inerythrocyte ghosts can be readily separated, by centrifugation, from thesoluble radioactive glycopeptide products to thereby facilitate theassay.

The proteolytic activity of P. haemolytica A1 culture supernatants,against ³ H-sialylated human erythrocyte ghosts, can be increased bydialysis of the supernatants, and by precipitation of an active proteinfraction at pH 4.5 (Table 1). The increased specific activity is duepartly to the removal of dialyzable inhibitors, and in part to theremoval of inactive proteins in the pH 4.5 supernatant. The previouslyreferred to leukotoxin of P. haemolytica A1 remains entirely in thesupernatant while the protease is quantitatively precipitated. Aseventeen-fold increase in the specific activity of the protease, andthe removal of leukotoxin, is achieved in the pH 4.5 precipitation step.This precipitate can be readily redissolved in 0.05M HEPES buffer pH 7.4and can be used in further studies for characterizing properties of theprotease because of the its high stability compared with fractions thathave lower protein concentrations.

Purification of the glycoprotease from serum-free culture supernatantwas achieved by size-exclusion and ion-exchange column chromatography asshown in Table 2. Separation of the neuraminidase and proteaseactivities was obtained when serum-free culture supernatants of P.haemolytica were concentrated by Amicon ultrafiltration andchromatographed by HPLC on TSK gel-exclusion colum. FIG. 10 shows thatthe neuraminidase is eluted in the void volume, but theglycophorin-degrading protease eluted mainly in a smaller M fraction.This protease-enriched fraction was chromatographed by HPLC on a MonoQanion exchange column, and the protease eluted with the major proteinpeak (FIG. 11). The highest specific activity of the protease in a majorfraction was 94 nmol per mg/h for glycophorin degradation, which was a188-fold increase over the activity of the serum-freeculture-supernatant, and recovery of the enzyme activity in thisfraction was 80%. SDS-PAGE analysis of this fraction showed a major bandat about 35,000 M_(T) and this fraction cleaved glycophorin A (FIG. 11inset). The isolation of a relatively homogeneous protein fraction witha glycoprotease activity similar to that found in the culturesupernatant, suggests that the activity is a discrete enzyme and thatmultiple factors are not involved in the hydrolysis of glycophorin. Noproteolysis was observed for chromatographic fractions other that thosecontaining the 35 kD protein. However, protease activity was veryunstable in fractions prepared from serum-free culture supernatants,compared with fractions containing serum proteins. Therefore, the stableactivity of pH 4.5-precipitated extracts are preferred as proteaseisolates for the enzyme.

Substrate specificity:

Glycophorin A is a 31 kD human erythrocyte membrane glycoprotein andcannot be the physiological substrate for the P. haemolytica A1protease.

However, few glycoproteins from bovine target cells are characterizedand available for testing as potential in vivo substrates. When N-linkedglycoproteins (immunoglobulins A1 and A2; bovine α-1 acid glycoprotein;bovine β-lactalbumin; and hen ovalbumin) were radiolabelled with ¹²⁵I-iodine and incubated with partially-purified enzyme extracts (pH)4.5-precipitated extract), no hydrolysis of these substrates could bedetected, SDS-PAGE and autoradiography. The lack of hydrolysis for humanimmunoglobulin A1 (IgA1) or human immunoglobulin A2 (IgA2), means thatthe glcyophorindegrading enzyme is not identical to IgA protease, amicrobial neutral metallo-protease (Labib, R. S., N. J. Calvanico, andT. B. Tomasi Jr. 1978. Studies on Extracellular Proteases ofStreptococcus sanguis, Purification and Characterization of a Human IgA1Specific Protease. Biochem. Biophys. Acta. 526:547-559, Plaut, A. G.1983. The IgA1 Proteases of Pathogenic Bacteria. Ann. Rev. Microbiol.37:603-622). A wide range of non-glycosylated proteins have been testedand found not be cleaved by the P. haemolytica A1 protease. The [¹²⁵I]-labelled proteins, bovine serum albumin, rabbit muscleglyceraldehyde-3-phosphate dehydrogenase, soybean trypsin inhibitor,bovine carbonic anhydrase, bovine trypsinogen and bovinechymotrypsinogen were not hydrolyzed by the protease. Other proteinswere tested as substrates by incubation with active enzyme fractions,and enzyme action was monitored by SDS-PAGE and Coomassie blue stainingof the substrate and products. The enzyme did not hydrolyze bovineinsulin chain A, insulin chain B, or cytochrome c. Partially purifiedenzyme preparations with high activity against glycophorin were inactivein cleavage of dye-casein conjugates (Azocasein) or dye-collagenconjugates (Azocoll). Thus the weak casein-degrading activity reportedin culture supernatants of P. haemolytica A1 (Otulakowski et al supra)was not found in the protease-enriched extracts used here. A number ofsmall synthetic peptides, including substrates cleaved by bacterialserine proteases, thiol proteases and neutral metallo-proteases, havebeen tested as substrates for the P. haemolytica A1 protease but nohydrolysis of small peptides was observed. No inhibition ofglycoprotease could be detected in the presence of conventionalinhibitors of serine proteases (phenylmethanesulfonyl-fluoride ordi-isopropylphosphofluoridate); thiol proteases (N-ethylmaleimide,p-chloromercuribenzoate or p-hydroxymercuribenzoate); chymotrypsininhibitor (tosylphenylalanyl chloromethylketone) or trypsin inhibitor(tosyl-lysl chloromethylketone).

Properties of the protease:

The protease activity was stable at pH 7.0-7.5, but was destroyedrapidly above pH 8.0. The protease is inhibited by prolonged treatment(24 h) with 1,10-phenanthroline, so that it has been classified as aneutral metallo-protease. Table 3 shows that the hydrolysis of [¹²⁵I]-glycophorin is inhibited completely by 1 mM EDTA, in serum-freepreparations of the enzyme, though more EDTA must be used to inhibit theenzyme if serum proteins are present, as for the pH 4.5 preparation(FIG. 9, lane e). However, dialysis against de-ionized (Milli-Q treated)distilled water to remove EDTA resulted in the reactivation of theenzyme, so that any metal ion activator must be tightly bound to theenzyme and not readily removed by EDTA (FIG. 9, lane f). TheEDTA-inhibited P. haemolytica A1 protease activity could not bereactivated by the addition of metal ions. In assays not shown here, theaddition of the divalent metal ions, Zn²⁺, Fe²⁺, Mg²⁺ or Ca²⁺ failed torestore the protease activity after EDTA treatment and dialysis of theenzyme, and failed to increase activity in the native enzyme. The anionscitrate and ascorbate gave similar inhibitions of the serum-free enzymeto that given by EDTA. The inhibition probably arises from masking bythe anions of tightly-bound metal ion activator on the enzyme, perhapsat some site which binds the anionic sialoglycoprotein substrates. Aputative zinc-binding site can be seen in the primary sequence of theenzyme as predicted from the nucleotide sequence of the gene of FIG. 4(SEQ ID NO:1). Free sealate did not inhibit the action of theglycoprotease liable 3). Phosphoramidon[N-(β-L-rhamnopyranosyl-oxyhydroxyphosphinyl)-L-leucyl-L-tryptophan] isa protein inhibitor of neutral metallo-proteases such as thermolysis, aprotease of broad specificity (Malfroy, B., and J.-C.Schwartz. 1985.Comparison of Dipeptidyl Carboxypeptidase and Endopeptidase Activitiesin the Three Enkiphalin-hydrolysing Metallopeptidases:"angiotensin-converting enzyme, thermolysin, and "enkephalinase".Biochem. Biophys. Res. Commun. 130:372-378, but without significanteffect on the P. haemolytica protease. Lack of inhibition byphosphoramidon is consistence with the inability of the enzyme tohydrolyze the thermolysin substrate, furoylacryloylglycyl-leucinamide(Otulakowski et al supra). The protease is not as heat-stable as isthermolysin. A 30 min preincubation of the enzyme at 40° C. gave rise to38% loss of activity in the presence of 25 mM NaCl, and to 80% loss ofactivity in the absence of 25 mM NaCl. Unlike thermolysin, the enzymewas entirely inactivated by 30 min pre-incubation at 60° C., in thepresence or absence of 25 mM NaCl.

Products of protease action on glycophorin:

The characterization of the products of protease action on glycophorinA, is technically difficult because the substrate and some products areover 60% carbohydrate, therefore these are highly soluble even in thepresence of protein denaturants, and they exhibit anomalouselectrophoretic behaviour (Futhmayr, H. 1978. Structural Comparison ofGlycophorins and Immunochemical Analysis of Genetic Variants. Nature(London) 271:519-524. The amphipathic nature of glcyophorin, a membraneprotein, means that the molecule is very susceptible to aggregation,even in the presence of detergents such as SDS (Segrest, J. P., I.Kahane, R. L. Jackson and V. T. Marchesi. 1973. Major Glycoprotein ofthe Human Erythrocyte Membrane: evidence for an amphipathic molecularstructure. Arch. Biochem. Biophys. 155:167-183). A dimeric substrate,cleaved at one site, will give rise to more than two fragments.Glycoproteins are also known to exhibit anomalous effects such asaggregation and poor SDS-binding which affect their apparent M_(T)values in chromatography and electrophoresis. Numerous attempts tosequence the major products, isolated by gel-exclusion chromatography orSDS-PAGE were frustrated by apparent N-terminal blockage or loss of theproduct in the first cycle of the Edman sequencing procedure. The sitesof cleavage of glycophorin A by the glycoprotease were identified byimmobilizing the enzyme onto nitrocellulose paper, with retention ofenzyme activity. Glycophorin A was incubated with the immobilized enzymefor 3 h and for 18 h, and the products were identified by appearance ofnew N-terminal sequences, corresponding to internal sequences of thesubstrate (Table 4). The first major product arises by the cleavage ofthe peptide bond at Arg31-Asp32 in glycophorin A, but further hydrolysisoccurs at other peptide bonds including Glu60-Arg61, Ala65-His66 andTyr34-Ala35. The major cleavage sites are not due to contaminatingproteases, since a similar pattern of product formation is seen forhomogeneous preparations of the enzyme on prolonged incubation withradio-iodinated glycophorin A. No N-terminal exopeptidase activity wasseen for the data of Table 4, as shown by the recovery of intactglycophorin N-terminal sequences. We have previously shown that theglycoprotease is not inhibited by a variety of trypsin and chymotrypsininhibitors (Otulakowski et al. supra). Furthermore, the major cleavagesof glycophorin A by trypsin and chymotrypsin are at Lys18 and Tyr20respectively, which is different from the major site of cleavage of theglycoprotease between Arg31-Asp32. (Tomita, M., H. Furthmayr and V. T.Marchesi. 1978. Primary Structure of Human Erythrocyte Glycophorin A.Isolation and Characterization of peptides and Complete Amino AcidSequence. Biochemistry 17:4756-4770, Tomita, M., and V. T. Marchesi.1975. Amino Acid Sequence and Oligosaccharide Attachment Sites of HumanErythrocyte Glycophorin. Proc. Natl. Acad. Sci. USA 72:2964-2968).

In order to understand the use of the glycoprotease and the discoveredDNA sequence, the following discussion identifies certain general areasof interest in the consequent use of the glycoprotease.

CD34 antibodies recognize primitive haematopoietic stem cells and theirleukaemic counterparts.

Four monoclonal antibodies MY10 Civin et al. 1984, J. Immunol, B1.3C5Katz et al. 1985. Leuk.Res., 12.8 Andrews et al. 1986. Blood and ICH3Watt et al. 1987. Leukaemia raised against KG1 or KG1a cells have beenshown to identify an antigen on a small population of bone marrow cells.This subpopulation is shown by colony-forming assays to includevirtually all unipotent (BFU-E, CFU-GM, CFU-Meg) and multipotent(CFU-GEMM and pre-CFU) progenitors Civin et al. 1984, J. Immunol.; Katzet al. 1985. Leuk.Res. MY10 has also been shown to bind to blastcolony-forming cells in cord blood Strauss et al. 1986. Exp. Hematol.While mature lymphoid colony forming cells do not express the CD34antigen, putative B lymphoid cell precursors with nuclear terminaldeoxynucleotidyl transferase activity also react with B1.3C5 and MY10Katz et al. 1985. Leuk.Res.; Strauss et al. 1986. Exp. Hematol.; Katz etal. 1986. Exp. Hematol. These and other studies, using fresh leukaemiasamples, indicate that the CD34-positive progenitor cells of normalmarrow contain precursors of myeloid cells Civin et al. 1984. J.Immunol.; Katz et al. 1985. Leuk. Res.; Andrews et al. 1986. Blood;Strauss et al. 1986. Exp. Hematol.; Katz et al. 1986. Leukocyte TypingII, Oxford Univ. Press and B cells Katz et al. 1985. Leuk. Res.; Katz etal. 1986. Leucocyte Typing II, Oxford Univ. Press; Ryan et al. 1986.Blood; Ryan et al. 1987. Blood. Recent analyses of rare T cell-ALLs withprimitive T-cell characteristics Watt et al. 1987. Leukaemia; Schuh etal. 1987. Blood and the undifferentiated T-cell leukaemia-derived celllines Molt-13 Watt et al. 1987. Leukaemia and RPM1 8402 Fackler et al.1990. J. Biol. Chem., strongly suggest that mature blood cells of alllineages are derived from the CD34-positive fraction of normal marrow.This is supported by the observation that CD34-positive bone marrowcells can reconstitute all lineages of the haematopoietic system inlethally-irradiated baboons. Animals receiving the CD34-negativefraction, failed to engraft Berenson et al. 1988. J. Biol. Chem. Apreliminary report of the transplant of CD34-positive cells in 3 womenwith metastatic breast cancer supports the view that isolatedCD34-positive marrow cells are also capable of reconstitutinghaematopoiesis in humans Berenson et al. 1988. Exp. Hematol. Recentsingle-cell cloning experiments demonstrate that highly purifiedCD34-positive cells, which lack co-expression of all myeloid T- orB-cell antigens, are capable of generating several types of colonieswhen grown over irradiated stromal cells in vitro Andrews et al. 1990,J. Exp. Med.

In healthy individuals, CD34 expression is confined topan-haematopoietic cells in the bone marrow, with the exception ofcapillary endothelial cells, which are CD34-positive on immunohistologicstaining Watt et al. 1987. Leukaemia; Beschomer et al. 1985. Am. j.Path.; Sutherland et al. 1986. Blood; Fina et al. 1990. Blood. Tumourexpression of CD34 tends to mirror normal expression. Essentially allnon-haematologic tumours are unreactive Watt et al. 1987. Leukaemia.About 40% of AMLs (mainly FAB M1 class) and 65% of cells are reactiveKatz et al. 1986 Leucocyte Typing II, Oxford Univ. Press whereas only1-5% of acute T-lymphoid leukaemia are reactive Civin et al. 1989.Leucocyte Typing IV, Oxford Univ. Press. Chronic leukaemia and lymphomasof more differentiated phenotype are uniformly negative Katz et al.1986. Leukocyte Typing II, Oxford Univ. Press; Civin et al. 1989.Leukocyte Typing IV, Oxford Univ. Press

CD34 is a highly glycosylated 110 kD molecule in leukaemic cells.

All CD34 antibodies recently assessed for the International Workshop onLeucocyte Differentiation Antigens, immunoprecipitate a monomericstructure of 110 kD from lysates of acute myelogenous leukaemia-derivedcell lines KG1 and KGla Civin et al. 1984. J. Immunol.; Katz et al.1985. Leuk. Res.; Andrews et al. 1986. Blood; Watt et al. 1987.Leukaemia. Similar bands can be isolated from fresh acute leukaemia ofprimitive myeloid, B-lymphoid and T-lymphoid phenotypes Katz et al.1985. Leuk. Res.; Schuh et al. 1990. Leukaemia (in press). Most CD34antibodies identify denaturation-resistant epitopes in western blots,though with widely different efficiencies Civin et al. 1989. LeucocyteTyping IV, Oxford Univ. Press. These antibodies recognize a variety ofdistinct epitopes on this antigen, some of which MY10, B1.3C5, 12.8, wehave shown to be dependent on the presence of sialic acid residues.Extensive structural and carbohydrate analyses indicated the presence ofO-linked glycans Fina et al. 1990. Blood; Sutherland et al. 1988.Leukaemia. Partial aminoacid sequence analysis has revealed nosimilarities with previously-described structures Sutherland et al.1988. Leukaemia. The CD34 cDNA has recently been cloned using amammalian expression system, COS-7 cells. It predicts that CD34 is atype I integral membrane protein of 40 kD, with 9 potentialN-glycosylation sites. Since the de-N-glycosylated and desialylatedforms are 90 kD and 150 kD respectively Sutherland et al. 1988.Leukaemia, the native molecule must contain a considerable number ofO-linked glycans. Accordingly over 35% of the amino acids in theN-terminal domain probably ensure that it takes on the conformation ofand extended rigid "pole". Thus the NH₂ -terminus of the CD34 antigencan be expected to extend a considerable distance out from the cellmembrane.

CD34 is a substrate for the P. haemolytica glycoprotease.

The progenitor-cell-restricted antigen CD34 on KG1 cells is readilycleaved by the P. haemolytica glycoprotease as shown by the loss ofreactivity of this antigen with the anti-CD34 monoclonal antibody B1.3C5Sutherland et al. 1990. Blood; Katz et al. 1985. Leuk. Res. whichdetects a sialic acid-dependent epitope on this glycoprotein Watt et al.1987. Leukaemia; and Sutherland et al. 1988. Leukaemia. We have assessedthe ability of this enzyme to remove CD34 epitopes recognized by allseven CD34 antibodies designated by the 4th Leucocyte Antigen WorkshopCivin et al. 1989. Leukocyte Typing IV, Oxford Univ. Press. We haveshown by fluorescence microscopy, quantitative flow cytometry andwestern blotting, that sialic acid-dependent epitopes, recognized byantibodies B1.3C5, MY10 Civin et al. 1984. J. Immunol. and 12.8 Andrewset la. 1986. Blood, are totally removed,as are the sialicacid-independent epitopes recognized by ICH3 Watt et al. 1987. Leukaemiaand 188.24 Sutherland et al. Leukaemia. In contrast, the sialicacid-independent epitopes by 115.2 Andrew et al. 1986. Blood and TUK3Civin et al. 1989. Leucocyte Typing IV, Oxford Univ. Press are totallyresistant to the action of the glycoprotease. As previously for thecleavage of glycophorin, the enzyme requires the presence of sialytedresidues on its substrate and the prior treatment of KGla cells withneuraminidase reduced the ability of the glycoprotease to cleave theICH3 and 188.24 epitopes Sutherland et al. 1990. Blood (in press). Thedifferential sensitivity of certain CD34 epitopes to cleavage witheither neuraminidase and/or glycoprotease establishes 3 classes ofepitopes:(Class 1) those like MY 10,B1.3C5 and 12.8 which are removedboth by neuraminidase and by the glycoprotease;(Class II) those likeICH3 and 188.24 which are removed only by the glycoprotease; (Class III)those like TUK3 and 115.2

which are removed by either enzyme. Polyclonal rabbit antibody will beraised against the P. haemolytica glycoprotease by injecting the pureenzyme expressed as the product of the recombinant gene.

For the study of the properties of the P. haemolytica glycoprotease, theenzyme used is the recombinant product in E. coli HB101 of the gene gpc,in the high-expression vector pTTQ18. The recombinant gene product issecreted into the periplasmic space of the host and has the advantage ofbeing free from P. haemolytica proteins and most host proteins. Theperiplasmic enzyme free from host proteins is isolated by osmotic-shock,followed by SDS-PAGE analysis. The enzyme band is clearly visible at 35kD by Coomassie staining and is cut from the gel and injected s.c. intorabbits. Rabbit sera is tested for specific antibody by western blottingagainst culture supernatant enzyme, with controls for host cell E. colilysates. These antibodies are used to make an immuno-affinity column bycrosslinking them to Protein A-Sepharose Schneider et al. 1982. J. Biol.Chem. The affinity column is used for the rapid isolation of the enzyme,both from the recombinant E. coli and from culture supernatants of P.haemolytica. The enzyme can be eluted from the affinity-column underconditions which give maximum recovery of active enzyme, as assessed bythe hydrolysis of radio-iodinated glycophorin A. The homogeneous nativeglycoprotease can be used to raise a series of monoclonal antibodieswhich can modulate the enzyme's activity. Neutralizing antibodies to theglycoprotease are needed to determine specific actions of the enzyme inaltering the structures and functions of cell-surface antigens.

Four cell-surface O-sialoglycoproteins, of known aminoacid sequence, arecleaved by the enzyme, and the products of cleavage isolated andcharacterized.

In our previous work, we have concentrated on the cleavage of humanerythrocyte glycophorin A by the P. haemolytica glycoprotease Udoh etal. 1986. Univ. of Guelph; Abdullah et al. 1988. Proc. Canad. Fed. Biol.Socs.; Abdullah et al. 1990. Biochem. Soc. Trans. This substrate iswell-characterized erythrocyte surface O-sialoglycoprotein and isreadily available for biochemical analysis. However, it is clear thatthe physiological targets of this enzyme are not erthrocyte antigens butare probably O-sialoglycoproteins on the surfaces of leukocytes orendothelial cells. SDS-PAGE analysis of immunoprecipitations performedwith specific monoclonal antibodies on lysates of surface-¹²⁵I-iodinated KG1a cells, a primitive myeloid/T-lymphois stem cell lineKoeffler et al. 1977. Science 200, have shown that theO-sialoglycoprotein cell surface antigens CD34 Civin et al. 1989.Leucocyte Typing IV, Oxford Univ. Press. CD43/leucosialin Killeen et al.1987. EMBO J.; Pallant et al. 1989. PNAS, CD44/lyaluronic acid receptorStamenkovic et al. 1989. Cell; Aruffo et al. 1990. Cell andCD45/leucocyte common antigen Thomas et al. 1989. Annu. REv. Immunol.;Pulido et al. 1989. J. Immunol. are all cleaved by the glycoprotease.Although the glycoprotease-sensitive structures contain some N-linkedglycans they are all extensively glycosylated with O-linkedcarbohydrates. However, the N-linked glycoproteins CD18/11^(a), 11^(b),11^(c) /leukocyte integrins Arnaout et al. 1990. Blood, CD71/transferrinreceptor Shae et al. 1986. Cell, HLA class I Owen et al. 1980. J. Biol.Chem., and 8A3 antigens Sutherland et al. Blood (in press, Jan. 1991),which lack O-sialo-residues, were resistant to the action of the enzyme.

O-sialoglycoproteins on cell surfaces have been implicated in a numberof roles in normal cells, and aberrant glycosylation has been detectedin a number of tumours Kanani et al. 1990. Can. Res. To confirm theutility of this enzyme in the analysis of glycoprotein structure andfunction of cell structure antigens, the list of susceptiblecell-surfaces can be readily extended. There are other blood cellO-sialoglycoproteins which would be substrates for the glycoprotease,including the T-cell associated antigens CD7 Sutherland et al. 1984. J.Immunol. and CD25/IL-2-receptor α chain Leonard et al. 1983. J.Immunol.; the pre-B cell associated antigens CD9 Newman et al. 1982.Biochim. Biophs. Acta and CD24 Piruccello et al. 1986. J. Immunol., andthe platelet/endothelial cell antigen, thrombomodulin Dirtman et al.1990. Blood. The latter glycoprotein is an endogenous anticoagulant, andhas potential uses in the control of cancer coagulopathies.Thrombomodulin bears some structural resemblance, in its domainorganization, to another membrane O-sialoglycoprotein, the low densitylipoprotein (LDL) receptor Russell et al. 1983. Cell.

To confirm that the enzyme is specific for O-linked glycoproteins, otherN-linked antigens can be tested as controls, including CD13 on KG1 andCD10 on pre-B cell lines.

The products of cleavage of the four O-sialoglycoprotein substrates areanalyzed by N-terminal sequencing, so that the susceptible peptide bondscan be identified.

The sites of cleavage of glycophorin A have shown that the glycoproteaseis unique. Its restricted substrate specificity for O-sialoglycoproteinsis shared by no other enzyme. The enzyme does not show thecharacteristic inhibitor or activator sensitivities of other knownprotease classes, for example, the serine proteases, thiol proteases,aspartate proteases or neutral metalloproteases Abdullah et al. 1988.Proc. Canad. Fed. Biol. Socs. The primary structure of theglycoprotease, predicted from the nucleotide sequence of the gene, showsno homology with other proteases of procaryotic or eucaryotic origin.The peptide bond specificity of the glycoprotease as further defined,permits study of structure-function relationships in substrateglycoproteins and cell-surface antigens. For example, we can use fiveO-sialoglycoprotein antigens, glycophorin A, CD34, CD44, CD45 andthrombomodulin, whose amino-acid sequences are known Tomita et al. 1975.PNAS; Sutherland et al. 1988. Leukaemia; Stamenkovic et al. 1989. Cell;Jackman et al. 1986. PNAS, and which are readily available. We haveshown that the first four antigens in this list are cleaved by theenzyme. Hydrolysis of glycophorin A can be carried out by theglycoprotease immobilized on nitrocellulose, which retains its activityand generates soluble products. The products can be isolated by SDS-PAGEunder conditions which prevent blockage of the new N-terminal sequencesMoos et al. 1989. J. Biol. Chem. and electrophoretically blotted ontoPVDF membranes (Immobilon) for N-terminal sequencing on a gas-phasemicrosequencer.

The analysis of the sites of glycoprotease cleavage of CD34 and CD44were performed on soluble CD34:IgG and CD44:IgG fusion proteins,chimeric molecules comprising the extracellular domains of either CD34or CD44 ligated to the Fc' domain of the human IgG₁ molecule. The fusionproteins are secreted into the medium to concentrations of 0.5 μg/ml byCOS-7 cells transfected with the appropriated genetically-engineeredplasmid Seed et al. 1987. PNAS and can be rapidly isolated from themedium by Protein A-Sepharose affinity chromatography. Purity isassessed by SDS-PAGE and silver staining. The purified CD34:IgG fusionprotein is analyzed by SDS-PAGE and western blots, with threesialate-dependent CD34 antibodies MY10, B1.3C5 and 12.8 (supra).Duplicate blots are neuraminidase-treated before additions of theantibodies. The fusion protein is cleaved with the P. haemolyticaglycoprotease and the products are chromatographed on ProteinA-Sepharose affinity columns, to remove N-terminal fragments. The boundmaterial is eluted and an aliquot analyzed by SDS-PAGE and silverstaining.

The approach described above for the determination of the site(s) ofcleavage of CD34, was also used to analyze the cleavage product for theCD44 molecule. Our experiments indicate that the 85kD cell-surface CD44molecule is split by the glycoprotease to yield two new bands of about40 kD and 50 kD, and suggest that there are two equally-susceptiblesites of cleavage in the CD44 molecule.

The use of glycoprotease to test the involvement of specificsialoglycoprotein antigens in transmembrane signalling during immunecell response.

As described above, the P. haemolytica glycoprotease can cleave a numberof O-sialoglycoproteins in the glycocalyx of living cells. The enzymedoes not show any cellular toxicity and does not cleave other proteinsand N-glycosylated proteins on the cell surface. Therefore, the enzymecan be a powerful new reagent with which to test current theories on theroles of O-sialoglycoproteins Jentoft et al. 1990. Trends in Biochem.Sci. It has been suggested that the primary purpose of O-glycosylationon the cell surface is to limit access to the plasma membrane bybacteria, viruses, toxins and cells of the immune system. A secondgeneral role for O-glycosylation may be the reduction of proteolysis andturnover for proteins which bear O-sialoglycosyl residues Kozarski etal. 1988. PNAS. In both cases, the high density of negative chargeconferred by sialate would inhibit the approach of other negativelycharged residues on other cells or enzymes. However, no matter whatgeneral effects may be attributed to the bulk of the O-glycoproteins oncell surfaces, there are indications of specialized biochemical rolesfor some cell surface O-glycoproteins. For example, CD43/leucosialin hasbeen implicated in transmembrane signalling Wong et al. 1990. J.Immunol. and the adhesion molecule, CD44, has been identified ashyaluronate receptor on the cell surface Aruffo et al. 1990. Cell. Theleukocyte common antigen, CD45, has an extracellular domain, thought tobe involved in ligand binding, and an intracellular domain with proteinphosphotyrosine phosphatase activity, so that it too may be atransmembrane signalling molecule, Thomas et al. 1989. Annu. Rev.Immunol. Other cell-surface O-sialoglycoproteins with defined functionsinclude the aforementioned LDL-receptor, the IL2-receptor-α-subunit CD25and thrombomodulin. Since the action of the P. haemolytica glycoproteaseappears to be restricted to this class of molecules, the enzyme can beused to confirm specific functions for known O-glycoproteins. The enzymecan also be use to diagnose the involvement of unknownO-sialogycoproteins in cellular response.

The role of cell surface O-sialoglycoproteins as cell adhesion moleculescan be tested by cleaving these with the P. haemolytica glycoproteaseand measuring the ability of the products to bind to specific ligands.

The CD44 antigen has been implicated in the specific homing ofleucocytes to specialized areas of endothelial cells which line thepost-capillary venules (High Endothelial Venule cells). Some monoclonalantibodies to this structure can specifically block this adhesion toHEVs Jalkanen et al. 1988. J. Immunol. The recent cloning of the cDNAfor CD44 confirmed the presence of N-linked and O-linked glycans andalso revealed the presence of four potential chondroitin sulfate linkagesites Stamenkovic et al. 1989. Cell. Sequence homology between CD44 andthe proteoglycan-link proteins suggested that CD44 may function as anextracellular matrix-binding adhesion molecule. This is supported by therecent finding that CD44 can bind with high affinity to hyaluronic acid,a component of the extracellular matrix Aruffo et al. 1990. Cell. TheCD44:IgG chimeric protein can be cleaved with P. haemolyticaglycoprotease and thereby assess the ability of the cleaved CD44:IgG tobind to hyaluronic acid. CD44 has also been shown to bind to othercomponents of the extracellular matrix, chondroitin-4-sulfate andchondroitin-6-sulfate, though with much lower affinity than that forhyaluronate Aruffo et al. 1990. Cell. We can now assess the ability ofthe cleaved CD44:IgG molecule to bind to these chondroitin sulfates.

The roles of O-sialoglycoproteins in cell activation can be tested inresponding cells from which cell surface molecules have been cleaved bythe P. haemolytica glycoprotease.

CD43/leucosialin is an O-sialoglycoprotein expressed in a variety ofglycosylated forms on human leucocytes. While its function is unknown,defective expression of the molecule has been associated with animmunodeficiency disease, the Wiskott-Aldrich syndrome Parkman et al.1981. Lancet 2; Greet et al. 1989. Biochem. Cell. Biol. Monoclonalantibodies against CD43 induce monocytedependent T-cell proliferation,and stimulation adhesion and hydrogen peroxide production in monocytes.Activation of T cells via anti-CD43 antibodies is associated withincreases in intracellular Ca²⁺, inositol phosphates and diacylglycerolWong et al. 1990. J. Immunol. We can now determine the effect of theglycoprotease on the intracellular increases in calcium induced by arabbit anti-CD43 antibody (gift from M. Fukuda, La Jolla, Calif.) inhuman PBL cells, loaded with the acetoxymethyl ester of INDO-1, afluorescent indicator of [Ca²⁺ ]_(i) Grynkiewicz et al. 1985. J. Biol.Chem.; Ebanks et al. 1989. Biochem J.

The progression of activated cells through the cell cycle as a result ofstimulation by lymphokines, will be tested in cells which have beentreated with the P. haemolytica glycoprotease to remove cell surfaceO-sialoglycoproteins.

T-cells can be stimulated to proliferate by mitogens and growth factorsthat are thought to act via O-sialoglycoprotein receptors or receptorcomplexes on the T-cell surface. We have used the murine T cell lineLBRM 331A which secretes IL-2 in response to mitogens, such as PHA, inthe presence of the lymphokine IL-1 and phorbol ester (TPA) Mills et al.1990. J. Cell. Physiol. Treatment of these cells with the P. haemolyticaglycoprotease concomitant with stimulation with mitogens for four hoursgave enhanced IL-2 secretion in the treated cells compared to controls.The glycoprotease preparation was not significantly mitogenic of itself,and was non-toxic to the treated cells. The data suggest that PHA andIL-1 do not stimulate cells via receptors that have O-sialoglycoproteinsstructures that are susceptible to hydrolysis by the enzyme under theseconditions The removal of O-sialoglycoproteins from T cell surfaces mayenhance the production of IL-2 in response to extracellular mitogens orIl-1. There may be increased binding of ligands to receptors, due to theremoval of some negatively charged glycopeptides on the cell surface.

The murine T cell line CTLL-2 proliferates in response to IL-2 and wasused to test the effect of the glycoprotease on IL-2 sensitivity. TheIL2-receptor x-subunit (CD25) is an O-sialoglycoprotein and may besusceptible to the enzyme, as measured by proliferation, over a widerange of IL-2 concentrations. Enzyme treatment alone was not mitogenicfor the CTLL-2 cells. By the use of mouse monoclonal antibodies anti-TACand 7G7.B6, which recognize different epitopes on the human IL-2R xchain, visualized by fluorescence microscopy using FITC-goat-anti-mouceIg, we can determine whether the enzyme treatment leads to any change inthe surface expression of the receptor. The enhanced sensitivity of theCTLL-2 cells to IL-2 may be due to unmasking of receptors by the removalof other O-glycoproteins of the glycocalyx. In control experiments thecell surface expression of another component of the IL-2 receptor, theI1-2-R β-subunit, can be monitored using the antibody DU2. Thispolyclonal antibody recognizes an extracellular domain and reveal anyunmasking of the receptor that is induced by enzyme treatment.

We can use the pre-B cell line BAF3-F7 to determine the effect of theenzyme on I1-2 and IL-3 receptors. This mouse cell line constitutivelyexpresses the I1-2R α-subunit and has been transfected with the gene forthe human I1-2R β-subunit, so that it will proliferate in the presenceof I1-2, and it can be stimulated by IL-3. Thus we can compare theenzyme effects on two lymphokine receptors in the same cell. We haveseen increases in sensitivities to both I1-2 and to IL-3 in these cellsafter treatment with the glycoprotease, with the more marked increasesbeing seen for IL-3 induced proliferation.

Some cell surface O-sialoglycoproteins are thought to be involved incell-cell interactions, and these interactions can be modified bycleaving such surface antigens with the glycoprotease enzyme. We cantest the effect of the enzyme on the in vitro MLR (mixed leucocytereaction), in which human peripheral blood leucocytes (PBL) from onedonor are irradiated and mixed with the non-irradiated PBLs of a seconddonor. The resultant cell-mediated proliferation over 72 h is monitoredby ³ H-thymidine incorporation. Enzyme-treated irradiated cells can beused with normal responder cells, and normal irradiated cell can be usedwith enzyme-treated responder cells. Glycoprotease treatment will be for30-60 min, and cells will be washed before mixing. Proliferation can bemeasured for all combinations of treated and untreated cells. The MLRexperiments should reveal whether the responder cells or theantigen-bearing irradiated cells are sensitive to glycoproteasetreatment.

The natural anticoagulant factor, thrombomodulin, found on humanplatelet and endothelial cells, will be solubilized by cleavage with P.haemolytica glycoprotease and soluble products will be tested for theirfunctionality in the clotting cascade.

Disseminated intravascular coagulopathy is a significant cause ofmortality and morbidity in cancer patients Dvorak et al. 1986. HumanPathol. The excessive clotting observed in these patients is caused byan overproduction of thrombin, and is very difficult to treat Dvorak etal. 1987 "In Haematosis and Thrombosis" ed. Maden, Hirsch et al.Thrombomodulin is a potent inhibitor of the blood clotting cascade andis found on the surface of platelet and endothelial cells. It forms a1:1 complex with thrombin, which converts Protein C to activated ProteinC, which in turn cleaves other activated coagulation cofactors.Thrombomodulin is type 1 transmembrane glycoprotein, with a proximalextracellular region rich in serine and theonine, potential sites of0-glycosylation. The domain organisation of thrombomodulin closelyresembles that of the LDL-receptor Jackman et al. 1986. PNAS. Suchorganization is thought to be crucial for receptor mediated endocytosisand supports the hypothesis that the function of thrombomodulin is toaid the internalization of thrombin. Soluble forms of thrombomodulin mayhave potential therapeutic use in the treatment of coagulationdisorders, such as DIC.

The following examples illustrate the cloning of the gene which encodesfor the glycoprotease in the development of antibodies thereto. Suchexamples are not intended to be limiting in any way to the scope of theinvention.

Material and Methods

Bacterial strains, plasmids and culture conditions:

E. coli strains HB101, TG-1, CSR603 and P. haemolytica A1 have beendescribed previously Lo et al. 1986. "A simple immunological detectionmethod for the direct screening of genes from clone banks." Can. J.Biochem. & Cell Biol; Lo et al. 1985. "Cloning and Expression of theLeukotoxin Gene of Pasteurella haemolytica A1 in Escherichia coli K-12."Infect. Immun.; Sancar et al. 1979. "A simple method for identificationof plasmid-coded proteins." J. Bacteriol. The preparation of a clonebank which contains the P. haemolytica A1 genomic DNA carried in thevector pBR 322 has also been described Lo et al. 1985. "Cloning andExpression of the Leukotoxin Gene of Pasteurella haemolytica A1 inEscherichia coli K-12." Infect. Immun. The E. coli recombinant clonesand in particular pPH1 and pPH8 which encode soluble antigens of P.haemolytica A1 were isolated using an antiserum directed against P.haemolytica soluble antigens in a colony immumoblot assay Lo et al.1986. "A simple immunological detection method for the direct screeningof genes from clone banks." Biochem. The M13 phage vectors mp18 and mp19and the expression vectors pTTQ18 and pTTQ19 were from PharmaciaChemicals Inc. (Dorval, QUE.). The E. coli HB101 clones were cultured inLT medium supplemented with ampicillin to 100 μg/ml (LT+A) Lo et al.1986. "A simple immunological detection method for the direct screeningof genes from clone banks." Can. J. Biochem. & Cell Biol. P. haemolyticacultures were grown in brain heart infusion broth (BHIB). E. coli TG-1was grown on Davis minimal medium Miller, J. H. 1972. "Experiments inMolecular Genetics." Cold Spring Harbor Laboratory.

Enzymes and chemicals:

All restriction endonucleases and DNA-modifying enzymes were fromBethesda Research Labs. (Burlington, ONT) or Pharmacia Chemicals Inc.and were used as recommended by the supplier. Goat anti-rabbitimmunoglobulin G-alkaline phosphatase conjugate, and colour developmentreagents, were purchased from Bio-Rad Labs. (Mississauga, ONT) or SigmaChemicals (St. Louis, Mo.) [α³² P]-dATP (3000 Ci/mmole) and Tran³⁵S-Label (1130 Ci/mmole) were from ICN Biochemicals (St. Laurent, QUE)and Immobilon™ polyvinylidene diflouride (PVDF) membranes were fromMillipore (Mississauga, ONT).

EXAMPLE 1

Screening for glycoprotease activity and enzyme assay:

The E. coli recombinant clones were grown in LT+A broth overnight,subcultured (1/100) and grown for 4 h at 37° C. to logarithmic phase.The cells were harvested by centrifugation (5000×g), washed in 50 mMN-2-hydroxyethylpiperazine-N¹ -ethanesulfonic acid (HEPES) buffer (pH7.4) and lysed by passing through a French press three times at apressure of 17,000 psi. The lysates were centrifuged at 1,085×g for 5min. to remove cellular debris and the supernatant, 0.1 ml was incubatedwith 5 μg ¹²⁵ I-glycophorin A (20 μCi/mg), prepared as described(10,11), in 0.1 ml 50 mM HEPES buffer (pH 7.4) for 16 h at 37° C. Theunhydrolysed substrate and products of hydrolysis were separated bysodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE)Laemmli, U.K. 1970. "Cleavage of structural proteins during the assemblyof the head of bacteriophage T4." Nature and detected by autoradiographyusing Cronex 4 X-ray film (Dupont, Wilmington DE). The enzyme activityof the glycoprotease was calculated from the percentage disappearance ofglycophorin A bands as determined by slicing the gel and counting theband in a gamma counter.

EXAMPLE 2

In vivo labelling of cloned glycoprotease:

The recombinant plasmids which included pPH1 were transformed in E. coliCSR603 and maxi-cells were prepared Sancar et al. 1979. "A simple methodfor identification of plasmid-coded proteins." J. Bacteriol. E. coliCSR603 carrying the recombinant plasmids were grown to mid-logarithmicphase at 37° C. in Davis minimal medium supplemented with ampicillin and0.5% caseamino acids. Then 10 ml of the culture was irradiated for 15sec at 400 μW/cm² using a G-E germicidal lamp (General Electric). Afterculturing for 2 h, 100 μl D-cycloserine (2 mg/ml) was added and theculture was grown overnight. Approximately 3 ml of the culture wascentrifuged and the washed pellet resuspended in 0.75 ml Davis minimalmedium supplemented with theonine (10 mg/ml), argine (15 mg/ml), leucine(15 mg/ml) and proline (10 mg/ml). The cell suspension was incubated for1 h at 37° C., after which 25 μCi of Tran ³⁵ S-Label was added. Forrecombinants in the expression vectors pTTQ18 or pTTQ19,isopropyl-β-D-thiogalactoside (IPTG, 0.5 mM) was included for inductionof the tac promoter. After labelling for 1 h, the cells were harvestedin a microfuge and lysed by resuspension in 150 μl of SDS-sample bufferLaemmli, U. K. 1970. "Cleavage of structural proteins during theassembly of the head of bacteriophage T4." Nature. The labelled proteinswere separated by SDS-PAGE according to Laemmli, U. K. 1970. "Cleavageof structural proteins during the assembly of the head of bacteriophageT4." Nature and identified by direct autoradiography of the dried gel.

EXAMPLE 3

Subcloning and DNA sequencing:

The 3.3 kbp BamHI-BgIII fragment of FIG. 2 from pPH1 was subcloned intothe BamHI site of the expression vector pTTQ19. Selective fragments wereremoved from the insert DNA by digestion and religation with anendonuclease having a restriction site located on the insert DNA and oneon the multicloning site in pTTQ19. A series of subclones were createdeach containing only selected fragments of the original insert DNA forpPH1. Each subclone was then analyzed for the expression of theglycoprotease in the E. coli maxi-cell system. A 2.3 kbp BamHI-HindIIIfragment of FIG. 2 was determined to contain the glycoprotease gene.This fragment was subcloned into the M13 phage vectors mp18 and mp19which propagated in E. coli TG-1. The nucleotide sequence of the insertDNA was determined by the dideoxy-chain termination method as describedpreviously Dale, et al. 1985. "A rapid single-stranded cloning strategyfor producing a sequential series of overlapping clones for use in DNAsequencing: application to sequencing the corn mitochondria 18s rDNA."Plasmid. The method of Dale was used to generate overlapping deletionsof the insert DNA in either M13, mp18 or mp19 vectors for sequencing ofthe whole insert DNA. The DNA sequence was analyzed by using the PustellSequence Analysis program (IBI, Toronto, ONT). The nucleotide data baseGenbank® was screened against the coding sequence, to search forsequence homology.

EXAMPLE 4

Expression of the glycoprotease (SEQ ID NO:1) gene:

The 2.3 kbp BamHI-HindIII fragment from pPH1 was subcloned into theexpression vector pTTQ18 to form the plasmid pGP1. Expression wascarried out by transformation of E. coli HB101 with pGP1. Cultures weregrown overnight at 37° C. in LT+A broth. The cultures were thenharvested by centrifugation (5,000×g) and the pellet was resuspendedinto prewarmed LT medium (without glucose) supplemented with ampicillinand IPTG (0.5 mM) and grown for 90 min at 37° C. The culture wascentrifuged 5,000×g) and the pellet resuspended in 0.1 vol. of 2X sodiumdodecyl sulfate (SDS)-sample buffer Lemmli, U. K. 1970. "Cleavage ofstructural proteins during the assembly of the head of bacteriophageT4". Nature. After boiling for 5 min the samples were separated bySDS-PAGE. The proteins were visualized by staining with Coomassiebrilliant blue R250. For the determination of glycoprotease activity,the cells were lysed by French press and the lysate assayed for activityas described above.

EXAMPLE 5

Preparation of antiserum against glycoprotease.

The glycoprotease expressed from pGPl was separated by preparativeSDS-PAGE and stained with Coomassie brilliant blue R250. The gelfragment containing the glycoprotease was excised, homogenized in 1 mlsaline containing the adjuvant saponin (Quil-A, Superfos a/s, Vedbek,Denmark) to yield an inoculum of 50 μg of Quil-A per rabbit. This wasinjected subcutaneously into two Pasteurella multocida-free New Zealandwhite rabbits (Hazelton Research Products Inc., Denver, Col.) at twoweek intervals. Serum was collected one week after the second injectionand tested for glycoproteaseneutralizing activity. The serum used forWestern-immuno blot analysis had titre of 1/100.

EXAMPLE 6

N-terminal amino acid analysis:

The glycoprotease expressed from pGPl was recovered after SDS-PAGE andtransferred by electroblotting onto PVDF membrane Walsh et al. 1988."Extended N-terminal sequencing of proteins of Archaebacterial Ribosomeblotted from two-dimensional gels onto glass fibre and PVDF membrane."Biochemistry. The PVDF membrane was stained with Coomassie blue R250, tolocated the gene product, destained and the appropriated region carryingthe glycoprotease was sliced out. The glycoprotease was then subjectedto N-terminal amino acid analysis by the automated Edman procedure in agas-phase peptide microsequencer.

The following examples provide a preferred methodology for isolating theglycoprotease from culture of a selected suitable serotype of P.haemolytica and its purification.

In addition, examples are provided of further characteristics of theglycoprotease.

MATERIALS AND METHODS

Bacteria and culture conditions: P. haemolytica A1 (biotype A, serotypei was originally obtained from E. L. Biberstein, University ofCalifornia, Davis, and is available under ATCC accession No. 43270.Stock organisms wer stored as lyophilized cultures after freeze-dryingin distilled water containing 5% (w/v) destran, M_(T) 70,000; 7% (w/v)sucrose and 1% (w/v) monosodium glutamate. Lyophilized P. haemolyticaserotype 1 was inoculated into a blood agar plate and incubated at 37°C. for 16-18 h. A few colonies were inoculated into 250 ml of brainheart infusion broth (BHIB) and incubated with shaking at 37° C. for 4.5h, the optimal time for enzyme production. The culture was centrifuged(8000×g) and the cells were resuspended into 500 mil of RPMI medium 1640(Gibco Laboratories, Grand Island, N.Y.) containing 7% heat-inactivatedfetal calf serum. For serum-free cultutre, only RPMI medium was used.The culture was grown for 3-4 h to midlog phase at 37° C. on a shaker,centrifuged (10,000×g) and the supernatant filter through a 0.2 μmMillipore filter. The filtrate was dialyzed against distilled water for48 h at 4° C. and lyophilized.

Purification of the P. haemolytica A1 protease:

The lyophilized P. haemolytica A1 culture supernatant was dissolved indistilled water (30 mg/ml) and was acidified to pH 4.5, with 10.Macetate buffer pH 4.0, to give 6 mM acetate concentration. After 30 minat 4° C., the precipitate which formed was removed by centrifuagation at47,000×g for 10 min. The precipitate contained most of the proteolyticactivity and the supernatant contained all the leukotoxin activity. Theprecipitate was dissolved at 400 mg of lyophilized material per ml in0.05M HEPES (4-(2-hydroxyethyl)-1-piperazine-ethane sulfonate) buffer,pH 7.4, for some enzyme assays, and is referred to as the pH 4.5fraction. Isolation of a homogeneous glycoprotease activity was achievedby HPLC separation of a serum-free culture supernatant preparation.Serum-free culture supernatant of P. haemolytica was concentrated byAmicon PM-10 membrane ultrafiltration to yield 0.5 mg/ml protein and 100μg of this protein was separated by gel-permeation HPLC, on two 60 cmTSK G4000-SW columns in series (separation range 5 kD-1000 kD), byelution with 0.1M Tris-HCl buffer pH 7.4 at 1 ml per min. The activeglycoprotease fraction was re-chromatographed by anionexchange HPLC on aMonoQ HR 5/5 column, with a gradient of 0-0.5M NaCl eluted at i ml permin. This yielded a single peak of glycoprotease activity, which onSDS-PAGE analysis showed a protein band at M_(T) 35 kD.

Immobilization of the glycoprotease:

Three pieces of nitrocellulose paper 1 cm² were soaked in 50 mM HEPESbuffer, pH 7.4 for 10 min and transferred to a concentrated serum-freepreparation of the enzyme, with gentle shaking for 30 min at 4° C. Theimmobilized enzyme was blocked with 1% hen ovalbumin for 1 h and washedwith HEPES buffer for 30 min with five changes of buffer.

Characterization of the products of glycophorin hydrolysis by the P.haemolytica A1 glycoprotease:

Human erythrocyte ghosts were prepared from outdated pooled blood-bankhuman blood (Blumenfeld, O.O, P. M. Gallup, and T. H. Liao. 1972.Modification and Introduction of a Specific Radiolabel into theErythrocyte Membrane Sialoglycoproteins. Biochem. Biophs. Res. Commun.48:242-251) and glycophorin A was extracted (Marchesi, V. T. H.Furthmayr, and M. Tomita. 1976. The Red Cell Membrane. Ann. Rev.Biochem. 45:667-678. Glycophorin A was further purified bychromatography on an agarose-wheat germ lectin affinity column(Pharmacia). For some experiments the glycophorin A was radio-iodinatedby the method of Markwell (Marwell, M. A. K. 1982. A New Solid-StateReagent to Iodinate Proteins. I. Conditions for the Efficient Labellingof Antiserum. Anal. Biochem. 125:427-432. To determine the sites ofglycophorin cleavage by the glycoprotease, 70-180 μg of [₁₂₅I]-glycophorin A (60,000) c.p.m.) incubated in 0.5 ml 50 mM Hepes bufferpH 7.4 with 3 cm² of the immobilized enzyme on nitrocellulose, preparedas described above. Incubation was for 3 h and for 18 h at 37° C. Theextent of glycophorin hydrolysis was determined by SDS-PAGE analysis ofradiolabelled products. Sites of cleavage of the glycophorin weredetermined by N-terminal amino-acid sequence analysis of the lyophilizedproducts, by the Edman procedure on a gas phase microsequencer (HSCBiotechnology Centre, Toronto).

Electrophoresis:

Enzyme fractions from gel-filtration and ion-exchange chromatography,and products of the digestion of radio-labelled glycophorin by P.haemolytica A1 protease, were examined by SDS-PAGE (Laemmli, U. K. 1970.Cleavage of Structural Proteins During Assembly of the Head ofBacteriophage T4. Nature (London) 227:680-685). The gels were fixed with45% ethanol containing 5% glacial acetic acid, washed with water, andstained either with Coomassie blue R250, with silver-staining or with"Stains-all" (Biorad, Mississauga, ON). The latter stain shows thepresence of sialoglycoproteins by a blue colour; non-sialylated proteinsare stained red. The position of ³ H-sialyl-glycoproteins on the gelswas carried out by fluorography by soaking the gels in scintillatorfluid (En³ Hance; NEN Canada, Dorval, QU). For fluorography orautoradiography, the dried gels were overlaid with X-ray film (XOMat,Kodak, Toronto, ON) and kept at -70° C. for 6 days in the dark, beforedevelopment.

Enzyme assays:

The P. haemolytica A1 protease was assayed by the hyrolysis of [³H]-sialyl-glycproteins labelled in situ in human erythrocyte ghostmembranes (Blumenfeld et al. supra). Enzyme fractions were incubatedwith [³ H]-sialyl human erythrocyte ghost membrane (0.25 mg protein) in150 μl 0.1M HEPES buffer, pH 7.4 for one to four hours at 37° C. At theend of the incubation the erythocyte ghosts were sedimented bycentrifugation at 13,000×g for 4 min, and 50 μl supernatant was removedand counted for ³ H-content by liquid scintillation counting. Controlscontained enzyme fractions which had been heat-denatured at 100° C. for15 minutes. The enzyme activity was measured against [³H]-sialyl-glycophorin and expressed in terms of nmol sialate content ofthe sialoglycopeptides released. In other assays, isolated glycophorinand other proteins were radio-iodinated with Na¹²⁵ I by the method ofMarkwell (Markwell supra). The cleavage of the enzyme of ¹²⁵ I-labelledglycophorin A was measured by incubating 5 μg radio-labelledglycophorin, 20 μCi per mg, with enzyme in 0.2 ml 0.1M HEPES buffer, pH7.4 for one hour at 37° C. The radio-labelled products and uncleavedsubstrate was separated by SDS-PAGE, and cleavage was estimated byslicing the gels and counting the distribution of radioactivity in agamma counter. Neuraminidase activity was measured either by use of thefluoroenic substrate, 4-methylumbelliferyl-α-D-N-acetylneuraminate, orby cleavage of sialyllactose. Sialyllactose, 0.1 ml 10 mM, was incubatedwith 0.1 ml enzyme fraction in 0.1M HEPES buffer, pH 7.4, for 4 h at 37°C. The released sialate was assayed by the method of Warren (Tomita, M.,H. Furthmayr, and V. T. Marchesi. 1978. Primary Structure of HumanErythrocyte Glycophorin A. Isolation and characterization of peptidesand complete amino acid sequence. Biochemistry 17:4756-4770. Protein wasassayed by the method of Peterson (Peterson, G. L. 1977. Asimplification of the protein assay method of Lowry et al. which is moregenerally applicable. Anal. Biochem. 83:346-356.

                  TABLE 1                                                         ______________________________________                                        Proteolysis of [.sup.3 H]-sialoglycoprotein of erythrocyte ghosts,            compared to macrophase cytotoxicity, for culture supernatant                  fractions of P. haemolytica A1.                                                              Pro-   Proteolysis  Cyto-                                                     tein   cpm/mg protein/h                                                                           toxicity.sup.a                             Fraction       mg     (% recovery) %                                          ______________________________________                                        1.  Supernatant    241.5  7,000 ± 100                                                                           100.0 ± 2.6                                                     (100)                                               2.  Dialyzed supernatant                                                                         241.5  12,000 ± 300                                                                           82.0 ± 3.1                                                     (171)                                               3.  pH 4.5-precipitate of                                                                         43.6  126,000 ± 2800                                                                        0                                            (1)                   (306)                                               4.  pH 4.5-supernatant of                                                                        195.2   0           100 ± 3.6                               (1)                    (0)                                                ______________________________________                                         .sup.a Cytotoxicity was measured as the release of [.sup.51 Cr] chromate      from bovine pulmonary macrophages (19), mean ± S.E.M., n = 3.         

                  TABLE 2                                                         ______________________________________                                        Isolation of the glycoprotease from serum-free culture                        supernatant of P. haemolytica A1.                                                          Glycoprotease                                                                            Specific                                                       Total Total    Activity Purifi-                                                                             %                                               Protein                                                                             Activity (nmol/   cation                                                                              Recov-                                          (μg)                                                                             (nmol/h) mg/h)    fold  ery                                    ______________________________________                                        Ultrafiltration                                                                          100     0.050    0.5    --    100                                  Amicon PM-10                                                                  TSK G4000-SW                                                                             28      0.042    1.5     3    84                                   (Fraction 6)                                                                  Mono Q HR 5/5                                                                            0.43    0.040    94.0   188   80                                   (Fraction 24)                                                                 ______________________________________                                    

                  TABLE 3                                                         ______________________________________                                        Effect of inhibitors on the cleavage of soluble [.sup.125 I]-glycophorin      by the P. haemolytica Al glycoprotease.                                                    Concentration   Activity.sup.a                                   Inhibitor    (mM)            (%)                                              ______________________________________                                        None                         100                                              Phosphoramidon                                                                             0.9             95     ± 4                                    Sialate      2.5             99     ± 1                                    Citrate      5.2             45     ± 1                                    Ascorbate    11.4            60     ± 2                                    EDTA         0.1             95     ± 5                                    EDTA         1.0             0                                                ______________________________________                                         .sup.a The serumfree enzyme was preincubated with the inhibitor for 30 mi     at 37° C., pH 7.4, followed by addition of soluble [.sup.125           I]-glycophorin and further incubation for 1 h. The degradation of             glycophorin was assayed by SDSPAGE analysis and autoradiography. The          disappearance of glycophorin A dimer was measured by densitometry of the      autoradiographs and is expressed as a percentage of enzyme activity in th     absence of any inhibitor. Negative controls contained heatdenatured enzym     with each inhibitor tested. Mean ± SD (n = 2).                        

                  TABLE 4                                                         ______________________________________                                        Specificity of glycoprotease for peptide bond cleavage of                     glycophorin A.                                                                Site of cleavage.sup.a                                                        Time (b)                                                                              P.sub.1  P.sub.1.sup.'                                                                           Sequence.sup.b                                                                        Amount (pmol).sup.c                        ______________________________________                                         3      Arg31    Asp32     32-39     9.2                                      18      Glu60    Ary61     61-70   38                                         18      Arg31    Asp32     32-39   37                                         18      Ala65    His66     66-79   14                                         18      Tyr34    Ala35     35-40   12                                         ______________________________________                                         .sup.a P.sub.1 -P.sub.1 ' represents the amide bond cleaved.                  .sup.b Glycophorin A amino acid residues identified for new Nterminal         sequence.                                                                     .sup.c The amount of cleavage represents the pmol of each new Nterminal       sequence, measured for the first amino acid. At both times, the major         sequence found was that of the Nterminal region of glycophorin A, from        cleaved and uncleaved substrate, and was 37 pmol at 3 h and 93 pmol at 18     h.                                                                       

    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 3                                                  (2) INFORMATION FOR SEQ ID NO:1:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 1315 base pairs                                                   (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (ix) FEATURE:                                                                 (A) NAME/KEY: CDS                                                             (B) LOCATION: 141..1115                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                       GGATCCAAGAATATGAAAGCAAAGAGCTACCGAATCCTGAAAAACTGAAGTATGGCGAAC60                AATTCTAGTCGTACAGAGAATAATGTGAGGGGCGTTCTTCGCCCCTTTTGGTTTTCTAAC120               TTATTTTGACTTCTCCAACTATGCGAATTTTAGGTATTGAAACCTCTTGT170                         MetArgIleLeuGlyIleGluThrSerCys                                                1510                                                                          GATGAAACCGGTGTTGCCATTTATGATGAAGACAAAGGCTTAGTGGCA218                           AspGluThrGlyValAlaIleTyrAspGluAspLysGlyLeuValAla                              152025                                                                        AACCAGCTTTATAGCCAAATTGATATGCACGCCGATTACGGTGGCGTA266                           AsnGlnLeuTyrSerGlnIleAspMetHisAlaAspTyrGlyGlyVal                              303540                                                                        GTCCCTGAACTGGCTTCTCGAGACCATATCCGTAAAACGTTGCCACTA314                           ValProGluLeuAlaSerArgAspHisIleArgLysThrLeuProLeu                              455055                                                                        ATTCAAGAAGCCTTAAAAGAGGCCAATCTGCAACCCTCGGATATTGAC362                           IleGlnGluAlaLeuLysGluAlaAsnLeuGlnProSerAspIleAsp                              606570                                                                        GGCATTGCCTATACTGCCGGCCCAGGCTTGGTCGGGGCTTTATTGGTC410                           GlyIleAlaTyrThrAlaGlyProGlyLeuValGlyAlaLeuLeuVal                              75808590                                                                      GGCTCAACCATTGCCCGTTCGCTGGCTTATGCTTGGAATGTTCCGGCA458                           GlySerThrIleAlaArgSerLeuAlaTyrAlaTrpAsnValProAla                              95100105                                                                      TTGGGCGTTCACCATATGGAAGGGCATTTACTTGCCCCAATGTTGGAA506                           LeuGlyValHisHisMetGluGlyHisLeuLeuAlaProMetLeuGlu                              110115120                                                                     GAAAATGCCCCTGAATTTCCGTTTGTGGCATTATTGATTTCAGGTGGA554                           GluAsnAlaProGluPheProPheValAlaLeuLeuIleSerGlyGly                              125130135                                                                     CACACCCAACTGGTAAAAGTTGACGGCGTTGGGCAATACGAACTACTC602                           HisThrGlnLeuValLysValAspGlyValGlyGlnTyrGluLeuLeu                              140145150                                                                     GGGGAATCAATTGATGATGCTGCCGGTGAAGCCTTTGACAAAACAGGC650                           GlyGluSerIleAspAspAlaAlaGlyGluAlaPheAspLysThrGly                              155160165170                                                                  AAACTACTCGGTTTGGATTACCCTGCCGGTGTAGCGATGTCAAAATTA698                           LysLeuLeuGlyLeuAspTyrProAlaGlyValAlaMetSerLysLeu                              175180185                                                                     GCCGAATCCGGCACGCCAAATCGTTTTAAATTCCCTCGTCCAATGACC746                           AlaGluSerGlyThrProAsnArgPheLysPheProArgProMetThr                              190195200                                                                     GACAGACCGGGACTGGATTTCAGTTTCTCCGGTTTAAAAACCTTTGCT794                           AspArgProGlyLeuAspPheSerPheSerGlyLeuLysThrPheAla                              205210215                                                                     GCGAATACGATTAAAGCCAATCTTAATGAAAATGGTGAACTCGATGAG842                           AlaAsnThrIleLysAlaAsnLeuAsnGluAsnGlyGluLeuAspGlu                              220225230                                                                     CAAACCAAATGCGATATTGCCCACGCATTCCAACAAGCCGTGGTTGAT890                           GlnThrLysCysAspIleAlaHisAlaPheGlnGlnAlaValValAsp                              235240245250                                                                  ACTATTTTAATTAAATGCAAGCGAGCGTTAGAGCAAACCGGCTATAAA938                           ThrIleLeuIleLysCysLysArgAlaLeuGluGlnThrGlyTyrLys                              255260265                                                                     CGCTTAGTAATGGCAGGCGGCGTAAGTGCCAATAAACAATTACGAGCA986                           ArgLeuValMetAlaGlyGlyValSerAlaAsnLysGlnLeuArgAla                              270275280                                                                     GACCTTGCGGAAATGATGAAAAAATTAAAAGGCGAAGTATTCTACCCT1034                          AspLeuAlaGluMetMetLysLysLeuLysGlyGluValPheTyrPro                              285290295                                                                     CGCCCACAATTTTGCACTGACAACGGCGCAATGATTGCCTACACTGGC1082                          ArgProGlnPheCysThrAspAsnGlyAlaMetIleAlaTyrThrGly                              300305310                                                                     TTTCTTCGCTTAAAAACGATGAACAAACCGACTTAAGCATTAGCGTAAACCCC1135                     PheLeuArgLeuLysThrMetAsnLysProThr                                             315320325                                                                     GCTGGCTATGACCGAATTACCACCGATTAATTAACCTTTCAAGCGGTGAAATTTCTTGTT1195              AATTTTGCAAAAATTTAATCAAAAATAACCGCTTGCTATATGATAGATTAAATTTATGAA1255              TAATTATGTAATTAGCCTACCTCCGCACAGGAGCGTAGAAAACATATTCAAGCTGAATTC1315              (2) INFORMATION FOR SEQ ID NO:2:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 325 amino acids                                                   (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                       MetArgIleLeuGlyIleGluThrSerCysAspGluThrGlyValAla                              151015                                                                        IleTyrAspGluAspLysGlyLeuValAlaAsnGlnLeuTyrSerGln                              202530                                                                        IleAspMetHisAlaAspTyrGlyGlyValValProGluLeuAlaSer                              354045                                                                        ArgAspHisIleArgLysThrLeuProLeuIleGlnGluAlaLeuLys                              505560                                                                        GluAlaAsnLeuGlnProSerAspIleAspGlyIleAlaTyrThrAla                              65707580                                                                      GlyProGlyLeuValGlyAlaLeuLeuValGlySerThrIleAlaArg                              859095                                                                        SerLeuAlaTyrAlaTrpAsnValProAlaLeuGlyValHisHisMet                              100105110                                                                     GluGlyHisLeuLeuAlaProMetLeuGluGluAsnAlaProGluPhe                              115120125                                                                     ProPheValAlaLeuLeuIleSerGlyGlyHisThrGlnLeuValLys                              130135140                                                                     ValAspGlyValGlyGlnTyrGluLeuLeuGlyGluSerIleAspAsp                              145150155160                                                                  AlaAlaGlyGluAlaPheAspLysThrGlyLysLeuLeuGlyLeuAsp                              165170175                                                                     TyrProAlaGlyValAlaMetSerLysLeuAlaGluSerGlyThrPro                              180185190                                                                     AsnArgPheLysPheProArgProMetThrAspArgProGlyLeuAsp                              195200205                                                                     PheSerPheSerGlyLeuLysThrPheAlaAlaAsnThrIleLysAla                              210215220                                                                     AsnLeuAsnGluAsnGlyGluLeuAspGluGlnThrLysCysAspIle                              225230235240                                                                  AlaHisAlaPheGlnGlnAlaValValAspThrIleLeuIleLysCys                              245250255                                                                     LysArgAlaLeuGluGlnThrGlyTyrLysArgLeuValMetAlaGly                              260265270                                                                     GlyValSerAlaAsnLysGlnLeuArgAlaAspLeuAlaGluMetMet                              275280285                                                                     LysLysLeuLysGlyGluValPheTyrProArgProGlnPheCysThr                              290295300                                                                     AspAsnGlyAlaMetIleAlaTyrThrGlyPheLeuArgLeuLysThr                              305310315320                                                                  MetAsnLysProThr                                                               325                                                                           (2) INFORMATION FOR SEQ ID NO:3:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 342 amino acids                                                   (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                       MetArgValLeuGlyIleGluThrSerCysAspGluThrGlyIleAla                              151015                                                                        IleTyrAspAspGluLysGlyLeuLeuAlaAsnGlnLeuTyrSerGln                              202530                                                                        ValLysLeuHisAlaAspTyrGlyGlyValValProGluLeuAlaSer                              354045                                                                        ArgAspHisValArgLysThrValProLeuIleGlnAlaAlaLeuLys                              505560                                                                        GluSerGlyLeuThrAlaLysAspIleAspAlaValAlaTyrThrAla                              65707580                                                                      GlyProGlyLeuValGlyAlaLeuLeuValGlyAlaThrValGlyArg                              859095                                                                        SerLeuAlaPheAlaTrpAspValProAlaIleProValHisHisMet                              100105110                                                                     GluGlyHisLeuLeuAlaProMetLeuGluAspAsnProProGluPhe                              115120125                                                                     ProPheValAlaLeuLeuValSerGlyGlyHisThrGlnLeuIleSer                              130135140                                                                     ValThrGlyIleGlyGlnTyrGluLeuLeuGlyGluSerIleAspAsp                              145150155160                                                                  AlaAlaGlyGluAlaPheAspLysThrAlaLysLeuLeuGlyLeuAsp                              165170175                                                                     TyrProGlyGlyProLeuLeuSerLysMetAlaAlaGlnGlyThrAla                              180185190                                                                     GlyArgPheValPheProArgProMetThrAspArgProGlyLeuAsp                              195200205                                                                     PheSerPheSerGlyLeuLysThrPheAlaAlaAsnThrIleArgAsp                              210215220                                                                     AsnXaaXaaXaaXaaGlyXaaThrAspAspGlnThrArgAlaAspIle                              225230235240                                                                  AlaArgAlaPheGluAspAlaValValAspThrLeuMetIleLysCys                              245250255                                                                     LysArgAlaLeuAspGlnThrGlyPheLysArgLeuValMetAlaGly                              260265270                                                                     GlyValSerAlaAsnArgThrLeuArgAlaLysLeuAlaGluMetMet                              275280285                                                                     LysLysArgArgGlyGluValPheTyrAlaArgProGluPheCysThr                              290295300                                                                     AspAsnGlyAlaMetIleAlaTyrAlaGlyMetValArgPheLysAla                              305310315320                                                                  GlyAlaThrAlaAspLeuGlyValSerValArgProArgTrpProLeu                              325330335                                                                     AlaGluLeuProAlaAla                                                            340                                                                           __________________________________________________________________________

We claim:
 1. A purified DNA molecule comprising a DNA sequence encodinga glycoprotease having the amino acid sequence shown in SEQ ID NO:2. 2.A purified DNA molecule comprising nucleotides 175-1168 of SEQ ID NO:1,coding for a glycoprotease having a molecular weight of approximately35.2 kD.
 3. A purified DNA molecule comprising at least 18 nucleotidesencoding a fragment of a glycoprotease amino acid sequence, saidfragment being selected from at least 6 corresponding sequential aminoacid residues of said amino acid sequence of SEQ ID NO:
 1. 4. Arecombinant expression vector comprising a DNA molecule of claim
 1. 5. Ahost cell transformed with a recombinant cloning vector comprising a DNAmolecule of claim
 1. 6. A host cell of claim 9, wherein said host cellis selected from the group consisting of prokaryotic and eukaryoticcells, said DNA molecule being operatively linked to an expressioncontrol sequence so that said glycoprotease is expressed, saidexpression control sequence being selected from the group consisting ofsequences that control the expression of genes of prokaryotic oreucaryotic cells.
 7. A process of recombinantly producing aglycoprotease, comprising culturing the host cell of claim
 5. 8. Aprocess of recombinantly producing a glycoprotease, comprising culturinga host cell transformed with a recombinant expression vector comprisingthe DNA of claim
 2. 9. The process of claim 7, further comprisingseparating said glycoprotease from said culture medium and purifyingsaid glycoprotease.
 10. The process of claim 7, wherein said host cellis Escherichia coli.